Hormones, Brain and Behavior || Transgenic and Genetic Animal Models

85 Transgenic and Genetic Animal ModelsR A Keri and R E Siegel, Case Western Reserve University School of Medicine, Cleveland, OH, USA

� 2009 Elsevier Inc. All rights reserved.

Chapter Outline

85.1 Introduction 2674

85.1.1 General Principles of Mouse Genetics 2674

85.1.2 Mice as Models for Behavioral Phenotypes 2676

85.2 Random Insertion of Foreign DNA into the Murine Genome 2677

85.2.1 General Principles 2677

85.2.1.1 Production of founder animals 2678

85.2.1.2 Patterns of inheritance 2678

85.2.1.3 Genomic regulation of transgene expression 2679

85.2.1.4 Insertional mutagenesis 2681

85.2.2 Transgene Composition 2682

85.2.2.1 Reporter genes 2683

85.2.2.2 Indicator strains of mice 2684

85.2.2.3 In vivo imaging 2685

85.2.2.4 Protein overexpression 2686

85.2.2.5 Transgene-mediated gene silencing 2687

85.2.2.6 Cell-specific ablation and inactivation 2688

85.2.3 Inducible/Conditional Expression of Transgenes 2690

85.2.3.1 Ligand-regulated transgene expression 2690

85.2.3.2 Recombination-induced transgene expression 2691

85.3 Homologous Recombination of Foreign DNA into the Murine Genome 2692

85.3.1 General Principles 2693

85.3.1.1 Targeting vectors and gene disruption in ES cells 2694

85.3.1.2 Production of chimeric mice 2696

85.3.1.3 Generation of knockout animals and pedigree analysis 2697

85.3.2 Knockin Mice 2698

85.3.3 Conditional Gene Targeting 2699

85.4 Gene Trapping 2701

85.5 Genetic Environments 2701

85.5.1 Genetic Background and Phenotypes 2702

85.5.2 Hitchhiking or Passenger Genes 2702

85.6 Summary 2703

References 2703

Glossarychimeric mouse A mouse that has contributions of

cells from two different sources: a donor

blastocyst and an embryonic stem (ES)

cell line.

congenic strains Two strains of mice or rats that

are genetically identical except for one

chromosomal region.

floxed allele An allele that is flanked by loxP

sequences and is the target of Cre

recombinase.

hemiallele An allele on one chromatid that does

not have a counterpart on the sister

chromatid.

hypomorphic allele A mutant allele that conveys

partial loss of gene function, either by

2673

2674 Transgenic and Genetic Animal Models

reducing expression or by decreasing

activity of the encoded protein.

inbred strain A strain of animals where all

individuals are genetically identical except

for gender.

modifier gene A gene within the genetic

background of an animal that modifies the

extent or manifestation of a phenotype.

outbred strain A strain of animals where the

individuals are genetically similar, but not

identical.

reporter gene A foreign gene inserted into the

genome that can be readily detected with

imaging tools or assays of enzymatic activity.

toxigene A gene whose expression induces cell

death.

85.1 Introduction

Rats have historically been the model of choice forperforming behavioral studies in rodents; however,the past decade has observed the emergence of miceas an equally powerful tool. This is due to the devel-opment of an extensive array of tools for rapidly andprecisely manipulating the murine genome. Geneticmanipulation of mice began in the early twentiethcentury when Clarence Cook Little developed twoof the first inbred strains: DBA and C57BL (source ofthe C57BL/6 substrain, Mouse Genome Informatics(MGI)). From his studies, it became obvious thatdifferent strains had unique phenotypic characteris-tics with distinct disease susceptibilities that wereindependent of environmental influences. The ageof mouse genetics had begun. From here, the devel-opment of molecular biology techniques coupledwith advances in murine embryology fueled anunprecedented growth in murine genetics, resultingin the development of countless models of humandisease. In 2007, Mario Capecchi, Sir Martin J. Evans,and Oliver Smithies were awarded the Nobel prize inphysiology or medicine for their discoveries of prin-ciples for introducing specific gene modification inmice by the use of embryonic stem cells. Theirpioneering work opened new avenues for discerninggenetic contributions to specific phenotypes. It wasno longer necessary to search for animal models withspontaneous phenotypic changes. Rather, traits couldbe genetically dissected with tools akin to molecularscissors. In addition, the small size and short genera-tion time of mice made them an essential model for

human behavioral traits. In this chapter, we discussthe basic principles of mouse genetics as it relates tobehavioral research, outline technical approaches formanipulating the mouse genome, and conclude withan overview of the impact of genetic complexitywhen evaluating a phenotypic trait.

85.1.1 General Principles of MouseGenetics

Although mouse and human evolution divergedapproximately 75million years ago, only about 300murine genes lack a human counterpart and viceversa. With both genomes containing approximately25 000 protein-coding genes, their degree of diver-gence in gene composition is only �1% (Watersonet al., 2002). This extent of similarity reinforces thepostulate that mice are valuable models for exa-mining the genetic basis of human physiology anddisease. The genetic underpinnings of behavioraltraits have historically been identified using classicalgenetics, meaning that a change in a trait occurredand the gene was subsequently identified. The funda-mental nature of a heritable trait dictates that animalscan have dominant, recessive, or co-dominant modesof inheritance. By constructing appropriate breedingparadigms, phenotypes of interest can be identifiedthat arise due to polymorphisms or chemicallyinduced mutations. It is important here to distinguishbetween polymorphisms and mutations. Polymorph-isms are differences in alleles that occur in all indivi-duals within a strain, whereas mutations arise in anindividual and distinguish this animal from the restof its strain. Polymorphisms are always heritablebecause they exist in every cell in the body, includingthe germ line. In contrast, mutations can be somaticor germ line and only those in germ cells will bepassed to subsequent generations. Analysis of poly-morphisms and germ-line mutations involves the useof forward genetics, which entails identifying a traitand then determining which gene(s) contributes tothat phenotype. This contrasts with reverse geneticswhich involves selectively mutating a gene of interestand discerning the resultant phenotype. Together,both approaches have unveiled expected and unex-pected genes/proteins that contribute to hormoneaction and associated behaviors.

Forward genetics requires using two general typesof mice: inbred and outbred. All mice within an inbredstrain are genetically identical except for gender.They are produced by 20 generations of brother �sister or parent � offspring matings, are homozygous

Transgenic and Genetic Animal Models 2675

at all genetic loci, and do not reject organs trans-planted from one individual to another. Phenotypictraits remain constant from one generation to thenext unless they are influenced by environmentalfactors such as diet. Although consistent withina strain, different inbred strains can have highly dis-similar phenotypes. Based on these natural differences,inbred strains have been useful for establishing numer-ous disease models. For example, BTBRT+ tf/J micehave several characteristics of autism, including lowsocial approach, resistance to change in routine, anddeficits in reversal learning (Moy et al., 2007) that areindependent of early postnatal maternal environment(Yang et al., 2007). Comparing the genetic differencesbetween these mice and those that have high socia-bility (C57BL/6J) should be useful for identifyinggenetic contributions to the autism-like behaviors(Yang et al., 2007). In addition to the advantagesafforded by strain-specific differences in phenotypes,the consistency within a strain permits considerablerefinement of experimental paradigms. Inter-animalvariability is minimized, affording a greater ability toidentify statistically significant differences. Moreover,environmental differences among animals should beminimal within a laboratory, greatly reducing thenumber of required subjects for each analysis andincreasing the number of diagnostic tests that can beperformed. An important tool for the selection ofrelevant mouse strains is the Mouse Phenome Data-base, which is devoted to cataloging readily accessiblephenotypic data for inbred strains, listing their intrin-sic behavioral traits.

In contrast to inbred strains, outbred mice aregenetically similar, but not identical. The degree ofsimilarity can vary considerably depending on howthe strain was generated andmaintained. Furthermore,mice from different vendors can be highly variableeven though they have the same strain name. Whenproducing outbred mice, the breeding of closelyrelated animals is intentionally avoided to maximizeheterozygosity (Chia et al., 2005). Outbred strains gen-erally have higher fecundity and vigor and tend to bemore disease resistant than their inbred counterparts.Consequently, they are considerably less expensive touse than inbred mice. It has also been proposed thatoutbredmicemore accurately reflect human genotypicheterogeneity. For this reason, they have been usedextensively in toxicological and pharmacologicalresearch (Chia et al., 2005). However, the high cost ofincreased variability in experimental data and theintrinsic genetic drift of these strains can limit theirutility. To restrict genetic diversity, closed colonies can

be produced. In this scenario, outbred mice areobtained and a colony generated without the specificintent of in- or outbreeding. The colony is self-populating and no new animals are introduced. Thisrestricts allelic variability and can be useful in caseswhere a particular inbred, mutant-mouse model haslow survival or reproductive capacity.

Congenic mice are a variant of inbred strains: theyare genetically identical with the exception of a singlelocus. They provide valuable tools for identifying chro-mosomal regions that underlie phenotypic differencesbetween strains. By having a single chromosomal seg-ment (usually containing several genes) from one strainin the genetic background of another, one can deter-mine if that locus or unrelated loci on different chro-mosomes are responsible for a particular phenotype.Congenic strains are produced by repeatedly back-crossing (>10 generations) a donor inbred strain witha recipient or destination strain. The ultimate goal is toreplace a locus in the destination strain with that sameregion from the donor strain.With each generation, thepresence of the donor locus of interest is confirmedusing polymerase chain reaction (PCR) assays orSouthern blotting. Only mice with the donor segmentare used for each subsequent round of breeding. Oncecongenicmice are produced, the relative importance ofthe donor locus to the recipients’ phenotype can beassessed. This approach has been used to identify locithat contribute to the differences in exploratory behav-ior of C57BL/6 and 129S1/SvImJ mice (Abe et al.,2000). Similar methods have been used in rats to iden-tify loci that modulate alcohol consumption (Carret al., 2006).

In addition to their utility for identifying behavior-modifying loci, congenic mice aid in determiningwhether the manifestation of a behavioral phenotypein response to a specific genetic change is modulatedby genetic context/background (Dominguez-Salazaret al., 2004; Bilkei-Gorzo et al., 2004). For example,disrupting the estrogen receptor a (ERa) gene com-promises various male sexual behaviors (Wersingeret al., 1997), but the extent of the phenotype is highlyvariable and depends upon the genetic strain harbor-ing the null allele (Dominguez-Salazar et al., 2004).This indicates that other genes modulate the pheno-typic outcome and supports studies aimed at theiridentification by quantitative trait mapping. Suchapproaches can reveal new genes that modulatereproductive behaviors and may unveil novel physi-ological pathways.

Genetic contributions to a phenotype may bemonogenic (one gene) or polygenic (multiple genes).


Both can be identified using breeding paradigms andfollowing the inheritance of genetic markers. If a traitis monogenic, identification of a locus is relativelystraightforward. PCR-based markers that span thegenome can be used to assess linkage of the pheno-typic trait with those markers. Using a sizable breed-ing colony to segregate alleles, a locus containing arelatively small number of genes can be refined. Onceknown and predicted functions (i.e., gene ontology)of the candidate genes are used to narrow the listof potential contributors, the impact of these candi-dates can be directly evaluated using transgenic orknockout approaches. The same type of approachis used for polygenic or complex genetic traits.However, these are much more difficult to identifybecause the impact of each locus on the trait will vary.Mapping requires identification of quantitative traitloci (QTLs), which are regions of the genome thatmeasurably contribute to a quantifiable phenotype. Ifa trait involves many genes, each with modest con-tributions, identifying QTLs will be challenging andrequire very large breeding colonies (Willis-Owenand Flint, 2006). However, if a trait involves a smallnumber of genes with substantial contributions, theseloci may be delineated. As above, identifying the truemodulators of a trait requires further refinement ofthe list of candidate genes followed by manipulatingthose genes in the mouse.

Genetic tools provide a cyclical paradigm forstudying the genetic basis of behavior in mice. Onecan begin with the traditional approach of character-izing a behavioral phenotype in inbred or chemicallymutagenized mice without any prior knowledge ofthe contributing genes. Following extensive pheno-type assessment, molecular approaches can be usedto evaluate the genome (analysis of DNA sequence),proteome (analysis of proteins), or transcriptome(analysis of mRNA expression), with the goal ofidentifying candidate genes (Sousa et al., 2006).Such genes are proposed to play a role in the devel-opment of the phenotype; however, testing thesehypotheses mandates further experiments. Usinga focused genetic approach, candidate gene activitycan be altered by generating transgenic or knockoutmice. These new mice are then subjected to addi-tional behavioral assessments, thus completing thecircular path to evaluating the genetic underpinningsof the particular trait. Entry into this path can occurat any point and it is equally likely to begin with aknockout mouse with a novel behavioral trait as itis to start by analyzing a well-documented trait andthen discovering its genetic basis.

Two primary web-based resources provide infor-mation and access to mice with genetically basedbehavioral phenotypes. MGI is a searchable databasethat facilitates selecting established mouse modelswith a particular behavioral trait (e.g., aggression).If available, the mice can be ordered from TheJackson Laboratory. The second resource is Neuro-mice.org, a multicenter consortium, including theNeurogenomics Project (Northwestern University),the Neuroscience Mutagenesis Facility (The JacksonLaboratory), and the Neuromutagenesis Project(Tennessee Mouse Genome Consortium). ThisNIH-supported consortium produces freely avail-able, novel mutant-mouse models that impact ner-vous system function and behavior. Informationregarding their characterization is accessible throughthis site as well.

85.1.2 Mice as Models for BehavioralPhenotypes

Rats have been used extensively for studies evaluat-ing the neurological basis of behavior and its phar-macological manipulation due to the well-establishedanalytical tools of operant and cognitive function aswell as the size and durability of these animals. Ratsare also readily amenable to invasive techniques suchas catheterization and placement of cannulas (CryanandHolmes, 2005).While rats have provided consider-able insight into mechanisms of behavior, the capabil-ities tomanipulate the genome are far superior inmice.In addition, there are relatively few inbred rat strainsavailable and techniques for manipulating the ratembryo are inefficient. As a result, there has been aremarkable expansion of the use of mice in behavioralresearch. It is important to emphasize that mice are notsimply small rats; rather, they have unique species-specific behaviors. Hence, their use has necessitatedthe adaptation of many tests originally developed forrats as well as development of novel, mouse-specificparadigms (Crawley and Paylor, 1997).

Intrinsic behaviors of distinct mouse strains varyconsiderably. For example, BALB/c mice are moresensitive to stress than other inbred strains and theyhave exaggerated responses to anxiety and depression-inducing stressors (Griebel et al., 2000; Anismanet al., 2001). Baseline differences in intrinsic beha-viors can have a large impact on experimental out-comes. If a behavior is already pronounced in a strain,the impact of a genetic change may be difficult todetect. In contrast, it might be readily discernable in astrain with a lower basal activity. The basal and


maximal activities of potential strains should bedetermined prior to any analysis, including geneticstudies, to preclude such pitfalls. In this way, a strainwith moderate basal behaviors can be selected wherea theoretical improvement or impairment can bereadily observed (Crawley et al., 1997). Considerthe phenotypes of mice that are deficient for theserotonin transporter (5-HTT). C57BL/6 mice withthis mutation display anxiety-like behaviors but129S6 mice with the same mutation do not. In thiscase, the high level of anxiety already present in the129S6 genetic strain precludes measuring an increaseover an exaggerated baseline (Holmes et al., 2003). Ifthese studies had been limited to 129S6 mice, themisleading conclusion would be that 5-HTT doesnot regulate anxiety.

In some cases, differences between strains canresult from sensory or physical impairments ratherthan a true behavioral trait. Some strains of mice haveretinal degeneration or are deaf. These animalswould generate a phenotype that could be erroneouslyinterpreted as a behavioral trait if inappropriate testswere used. This concept is further exemplified by thewide variability in size of the corpus callosum amonginbred mouse strains. Of note, BTBR T+ tf/J, 129/J,and 129SvJ all have severe reductions in the size ofthe corpus callosum, making them intrinsically poorlearners (Balogh et al., 1999). The development of thisstructure is highly similar between humans and mice;thus, mice provide an excellent animal model fordetermining the genetic basis for differences in itsformation (Richards et al., 2004; Kusek et al., 2007).However, it is important to emphasize that genesregulating development of this structure cannot beconstrued as directly affecting behavior.

Phenotypic differences in strains could be viewedas a deterrent to using mice in behavioral studies.However, these distinctions are actually a strength ofmouse modeling because they reflect populationdifferences in humans. As indicated above, a uniquefeature of inbred mice is that all mice within a par-ticular strain are genetically identical, except for sex,which greatly facilitates identifying the genetic basisof a trait. The tryptophan hydroxylase 2 (Tph2) gene,which regulates neuronal synthesis of serotonin(5-HT), provides an illustrative example. The Tph2protein is polymorphic among mouse strains at resi-due 447. BALB/cJ and DBA/2 mice contain a prolineat this site while C57BL/6 and 129X1/SvJ mice havean arginine (Zhang et al., 2004). This leads to differ-ences in 5-HT synthesis in the frontal cortex andstriatum, with the proline residue corresponding to

lower levels of protein. This observation led to charac-terization of a similar loss-of-function polymorphismin the Tph2 gene in humans that may contributeto unipolar major depression (Zhang et al., 2005).This study demonstrates an important experimentalparadigm. Specifically, genetic differences in candidategenes in mice can be used as launching points fortargeted sequencing analyses of patients.

85.2 Random Insertion of ForeignDNA into the Murine Genome

Although the strain-specific differences describedabove have been useful for characterizing geneticcontributions to specific behaviors, their utility islimited by the random acquisition of informativepolymorphisms between strains. In contrast to thisstochastic process, technology has been developedin the last two decades that permits directed altera-tions of the murine genome. The first gene-specificmanipulations of the mouse involved inserting for-eign DNA (i.e., transgenes) into the genome, resultingin transgenic animals. This technology was developedin the early 1980s as a result of the tremendousconcomitant strides that had been made in recombi-nant DNA technology and murine embryology.

85.2.1 General Principles

Initial studies demonstrating that foreign DNA couldbe inserted into the mouse genome were focused ondetermining if the contrived genes would producefunctional proteins and be heritable (Gordon et al.,1980; Brinster et al., 1981; Constantini and Lacy, 1981;Wagner et al., 1981; Gordon and Ruddle, 1981). Onceheritability had been demonstrated, it became clearthat the power of this approach would rely on confin-ing expression of a transgene to select subsets of cellsrather than the entire organism. For example, studiesinvolving SV40 T-antigen expression in mice resultedin the formation of brain tumors as well as patholo-gies in the thymus and kidney (Brinster et al., 1984).The complex array of pathological changes observedwith global expression limited the ability to discerneffects of the transgene on individual tissues, leading tothe characterization of tissue-specific promoters thatconvey protein or RNA expression within specific celltypes. From that point, most transgenic mouse studieshave relied on this same conceptual framework wheretransgenes are used to express a selected protein orRNA in specific cell types.

TSP EC A +TSP EC A+

Donor

Pronuclearinjection

Recipient

Injected pups

+− − − − Genotype

(a)

(b)

I

Figure 1 (a) A typical transgene contains a tissue-

specific promoter (TSP), an expression cassette (EC),

an intron (I), and a polyadenylation signal (Aþ).(b) Standard pronuclear injection procedure.


85.2.1.1 Production of founder animals

Insertion of transgenes into the murine genome canbe accomplished through four main routes: pronu-clear injection of fertilized oocytes, transfection orviral infection of embryonic stem (ES) cells (Naganoet al., 2001; Pfeifer et al., 2002), sperm-mediatedtransgenesis (Lavitrano et al., 1989), or viral infectionof preimplantation embryos (van der Putten et al.,1985; Stewart et al., 1987). In addition, protocols haverecently been optimized for production of transgenicrats by pronuclear injection of DNA (Filipiak andSaunders, 2006), suggesting that this model mayeventually become as pervasive as transgenic mice.

The primary method for producing transgenicmice is injection of linearized DNA (i.e., the trans-gene) into the male pronucleus of fertilized oocytes(Figure 1). If the transgene is housed in a plasmidvector, the transgene is usually removed from thevector, rather than simply linearizing the DNA.Although the rate of transgene insertion into thegenome is unaffected by plasmid sequences, theplasmid may contain elements that suppress trans-gene expression (Townes et al., 1985; Schedl et al.,1992). Following injection, the embryos must beplaced into an appropriate uterine environment forembryonic and fetal development. The reproductivetract of the mother of the fertilized embryos isdestroyed during embryo collection; thus, a hormon-ally primed surrogate is used. This is accomplished

by mating with vasectomized males which generates apseudopregnancy in the surrogate. Once pups areborn, they are genotyped to reveal which haveincorporated the foreign DNA. The rate of transgen-esis depends on multiple factors, with the most criti-cal being the skill of the microinjector and the qualityof the isolated transgene DNA. Typically, 10–30% ofthe pups born will be transgenic founder animals.Each transgenic pup is the product of one injectedembryo and the subsequent integration of foreignDNA into a unique site in its genome.

Injected DNA integrates randomly and relativelyrarely; thus, most mice will harbor a transgene at justone site in their genome. In theory, every nucleotidein the genome (3 � 109 for mice) could serve as anintegration site for transgenic DNA. While a singlesite in the genome receives the transgenic DNA,many copies of the transgene can be inserted in thatsite. Mice can harbor just a single copy of the trans-gene or tandem arrays that range from two to a fewhundred copies due to the formation of concatemersprior to insertion (Bishop and Smith, 1989). Occa-sionally, the DNA can be integrated in multiple sites.Given the random nature of integration, each trans-genic pup represents a new mouse model with thetransgene being inserted in different regions. Theseanimals are termed founder or F0 mice. To genotypethese mice, a small piece of tissue (usually from thetail, toes, or ears) is removed and the DNA is isolated.The genomic DNA is then evaluated by Southernblots using a transgene-specific probe or PCR withtransgene-specific primers. These founders can beused immediately in experiments; however, usingthem to produce novel lines of mice provides twoadvantages. The most practical advantage is that moremice can be produced by breeding the founder,increasing the population of study subjects. Second,progeny from a single founder will all have the samenumber of copies of the transgene integrated into thesame site in the genome; hence, they are more likelyto generate consistent results. The exception to thistenet occurs when the founder animal has multipleintegrations of the transgene in different sites of thegenome. These will independently segregate in thegerm line, resulting in a mixed population of progeny.It is important to segregate these different integrantsthrough multiple rounds of breeding and genotypingto limit the potential of variable phenotypes.

85.2.1.2 Patterns of inheritance

Once founder mice are obtained and bred, eachfounder will generate offspring displaying one of


the four possible patterns of inheritance. These aredictated by the timing of DNA integration, the num-ber of integration events, and the potential for inte-gration into a sex chromosome. If the transgeneintegrates prior to the embryo’s first cell division, allcells in the resultant mouse, including the germ line,will contain the transgene. In this case, the transgenewill be inherited according to Mendelian genetics asa heterozygous allele, although technically it is ahemiallele because the sister chromatid does nothave a complimentary allele. If the DNA integratesinto one chromosomal locale, 50% of the progenyshould be transgenic. They can then be bred tohomozygosity or perpetually bred to nontransgenicmice to maintain the line. Importantly, two founderanimals should never be bred to one another becausethey have different integration sites, generatingunnecessary genetic complexity.

If injected DNA integrates after the first celldivision, a mosaic mouse will be produced with thetransgene present in only a subset of cells. In aseverely mosaic animal, the transgene may not bepresent in many germ cells. If so, the founder maynot pass on the transgene, or may only generate afew transgenic offspring. Importantly, if any trans-genic offspring are obtained, the F1 mice will havethe transgene in every cell. Hence, they can thenbe used to generate a line where the transgenewill be inherited as a hemizygous allele as describedabove. Mosaicism in the germ line is a reflectionof general mosaicism in the entire animal. Thus,severely mosaic animals are also inappropriate forphenotypic characterization because the cells or tis-sues of interest may contain only a few transgeniccells or none at all. These animals increase experi-mental variability. The percentage of mosaic founderanimals obtained from pronuclear injection is vari-able, but can be up to 62% (Whitelaw et al., 1993),indicating that most transgenes integrate after thefirst cell division. The extent of mosaicism withinan individual can be relatively minor and the cohortof progeny may be sufficiently small to precludeaccurately assessing inheritance patterns. A recenttechnical development has been used to overcomethe issue of mosaicism. This involves sperm-mediatedgene transfer coupled with intracytoplasmic sperminjection (ICSI) of oocytes. In this procedure, trans-gene DNA is incubatedwith frozen and thawed spermand individual sperm are then injected into oocytes.Although relatively underutilized, this techniqueresults in very low levels of mosaicism, with foundersgenerating transgenic progeny according to expected

Mendelian inheritance patterns (Perry et al., 2001;Moreira et al., 2007).

Another type of pedigree occurs when the trans-gene integrates into multiple sites in the genome,with each integration site being inherited as a hemi-allele. Most often, these integrations occur in differentchromosomes; thus, the alleles are inherited indepen-dently. The number of integration sites dictates thepercentage of positive progeny from a founder mouse.For example, two integration sites will give rise to�75% positive offspring, while three integrationsites will yield �88% positive progeny. The last ped-igree category arises if the transgene integrates intothe X chromosome. In a female founder, 50% of herprogeny will inherit the transgene. Her male trans-genic progeny will father only female transgenics.This same pattern will be obtained from a malefounder with an X chromosome insertion. Integrationinto the Y chromosome rarely happens due to itssmall size; if it does happen, only male transgenicmice can be obtained.

85.2.1.3 Genomic regulation of transgene

expression

As indicated above, integration of transgenic DNAinto the genome is random. Some insertions occur intranscriptionally silent regions of the genome whileothers are within or near actively transcribed genes.The nature of the surrounding genomic environmentcan have a large influence on the activity of a trans-gene. Indeed, the chromatin environment usuallyhas a greater influence on the absolute activity ofa transgene than the number of copies of the trans-gene that were integrated. Hence, transgene activityis said to be integration site-dependent and copynumber-independent.

A typical transgene contains a promoter regu-latory region that should convey appropriate expres-sion to the cell type of interest. This usually involvesthe use of a small region of a promoter with a fewdefined regulatory elements. The expression of allendogenous genes is dictated by a series of cis-actingtranscriptional regulatory elements known as theexpression domain. These elements may reside closeto the start site of transcription, several kilobasesaway, or within introns. For the most part, promotersused for transgenes lack the complete array of neces-sary sequences to guard against the regulatory controlof the surrounding chromatin. As a consequence,transgene expression is often affected by the particu-lar site into which it is integrated, leading to altera-tions in the absolute level, sites, and timing of


expression. This is known as position variegation.Variegation is due to two primary components of thegenomic neighborhood (Figure 2). One of these isinvasive heterochromatin, where densely compactedregions spread along the chromosome to includethe transgene. The most obvious example of thisis when transgenes integrate into regions of thegenome that consist of dense heterochromatin, suchas centromeres or telomeres, which are transcription-ally silent. In this case, the founder may producetransgenic offspring, but all progeny will be incapableof expressing the transgene. This is an extreme exam-ple involving gene deserts: areas of the genome thatlack genes. More often, transgenes integrate intoregions of the genome that have more varied expres-sion. Here, transgene expression will be under moremodest control from the surrounding environment bytranscriptional enhancer elements within adjacentgenes. These elements can also regulate neighboringtransgene activity. As a result of these two processes,expression of a transgene is dominated more by itsgenomic neighborhood than the number of copiesthat have integrated into the genome.

Neighboring chromatin has much less influenceon the expression of endogenous genes compared tointegrated transgenes because endogenous genes con-tain insulator or boundary elements. Insulators aredefined by two functions: (1) they block communica-tion between promoters and enhancers when placed

E

E B B

Open chromatin

Heterochromatin

Adjacent enhancer

Border element protection

Figure 2 Position variegation of transgene expression.

Depending on the integration site and presence/absence ofborder elements, a transgene can be highly expressed or

not expressed at all. Arrows indicate levels of expression.

between these two regulatory sequences, and (2) theyprotect transgenes from heterochromatin-mediatedgene silencing when placed on each end of the trans-gene (Recillas-Targa, 2006). Insulators do not intrin-sically activate or repress gene expression. Rather,they create expression domains and prevent expan-sion of heterochromatin into that domain. They alsoblock distant enhancers from modulating the activityof an insulated gene (Recillas-Targa, 2006). Engi-neering insulators onto each end of a transgene hasproven effective for preventing position effects, lead-ing to copy-number-dependent expression. Thechicken b-globin cHS4 insulator has been the mostcommonly used for this purpose (Potts et al., 2000).It should be noted, however, that addition of thisinsulator does not guarantee independence fromposition variegation, indicating that a greater under-standing of the functions of insulators is necessarybefore their use becomes routine.

In many instances, the purpose of producing atransgenic mouse is to assess the ability of a transgeneto complement a particular phenotype. Specifically,an allelic variant from one mouse strain may be hypo-thesized to underlie a recessive phenotype. Comple-menting the phenotype with a dominant allele fromanother strain would be the most straightforwardapproach to testing this possibility. Complementa-tion studies are only interpretable if the transgeneis expressed to a similar extent as the endogenousgene, and position variegation complicates this typeof analysis. This pitfall can be avoided by usingtransgenes that contain all of the transcriptionalregulatory elements for a particular gene. Expressionof such transgenes would be position-independentand copy-number-dependent. Unfortunately, the fullarray of regulatory elements is not known for mostgenes and identifying them would require a resource-intensive detour prior to completing the desired exper-iment. This hurdle can be overcome with the use ofgenomic-type constructs, which contain very largeregions of genomic DNA (Schedl et al., 1992, 1993;Lamb et al., 1993; Strauss et al., 1993; Jakobovits et al.,1993). These can be bacterial (BAC), P1 (PAC), oryeast (YAC) artificial chromosomes, ranging in sizefrom 100Kb to more than 1Mb. The large size virtu-ally assures that the transgene contains all of thenecessary regulatory elements to convey expressionwith the same spatial and temporal specificity as theendogenous gene, hence recapitulating its expressiondomain (Giraldo and Montoliu, 2001).

AYAC transgenic approach has been used to over-express the human amyloid precursor protein (APP)


in the brain to determine if increasing the levels ofeither wild-type or mutant protein would inducefeatures of Alzheimer’s disease in mice. Employmentof a YAC vector was necessary because previousattempts to target APP overexpression to thebrain with conventional transgenesis resulted in verylow expression (Lamb et al., 1993). The APP YACtransgenes, in combination with a presenilin 1 YACtransgene, resulted in deposition of amyloid that wasdependent on the APP transgene copy number (Lambet al., 1999). This outcome confirmed that the levelof APP expression is a major determinant of peptidedeposition. YAC transgenic mice have also been usedto assess the impact of a polyglutamine track inthe androgen receptor (AR). It has been suggestedthat expansion of this track contributes to X-linkedspinal and bulbar muscular atrophy (SBMA), a formof neuromuscular degeneration. Using YAC trans-genes ensured appropriate expression in the CNSand revealed that expression of an AR with varyingnumbers of glutamines induces progressive neurode-generation in mice similar to what is observed inhumans (Sopher et al., 2004).

In addition to the effects of surrounding chroma-tin, the mere presence of several copies of the trans-gene in a tandem array can silence its activity.Repeat-induced gene silencing was demonstratedusing a Cre/ lox transgenic mous e model (see Secti on85.2.3.2) in which the numb er of trans gene re peatscould be decreased within the same chromosomallocation (Garrick et al., 1998). This approach elimi-nated position variegation, permitting direct assess-ment of the number of transgene repeats onexpression levels. This study revealed that reducingtransgene copy number actually increases its expres-sion, indicating that concatemerization of the trans-gene negatively affects its transcription. Hence, as ageneral rule of thumb, selection of individual lines oftransgenic mice to be used in a study should bedictated by the temporal, spatial, and absolute levelsof expression rather than copy number.

As indicated above, genomic type constructs (i.e.,artificial chromosomes) are randomly integrated, butthey are shielded from the impact of surroundingchromatin due to the presence of intrinsic insulators.In contrast to this approach, a transcriptionally activeregion of the genome can be preselected for targetedintegration of the transgene employing homologousrecombin ation (see Section 85.3; Bro nson et al., 1996;Cvetkovic et al., 2000; Evans et al., 2000). This avoidsthe quelling effects of heterochromatin and a singlecopy can be inserted, preventing repeat-induced

silencing. This is an ideal method when it is necessaryto obtain the same level of expression with multipletransgenes. For example, when comparing the func-tion of two protein isoforms, it is necessary that theirexpression is at the same level in all tissues.

A highly effective site for integrating transgenesis the ROSA26 [Gt(ROSA)26Sor] locus. Origi-nally identified by a gene-trap study (Friedrich andSoriano, 1991), the ROSA26 locus is transcription-ally active in all tissues during embryogenesis(Zambrowicz et al., 1997), presumably due to a uni-formlyopen chromatin configuration.Ab-galactosidasereporter transgene integrated into this site is robustlyexpressed in all tissues, providing an advantageousmodel for tagging cells in chimeric animals as well astissue-recombination studies (Zambrowicz et al., 1997;Lo et al., 2004). Equally important, the ROSA26 locushas been used as a recipient site for insertion of othertransgenes, leading to more uniform expression.Of note, the orientation of the transgene in theROSA26 locus and the presence/absence of a select-able marker can impact its absolute expression(Strathdee et al., 2006). Hence, it is essential that thecomposition and orientation of all transgenes remainconstant when using this approach.

85.2.1.4 Insertional mutagenesisThe stochastic nature of transgene insertion meansthat it may integrate into an endogenous protein-coding gene, disrupting its function. Only about 3%of the mouse genome is comprised of protein-codinggenes, making this a relatively rare event. However,non-protein-coding genes and regulators of chroma-tin configuration can also be targets for integration,increasing the probability of integrating into a criticalgenomic site. Combined, it is estimated that 5–10%of all transgenic lines generate an obvious phenotypedue to disruption of an important genomic element(Meisler, 1992). This typically goes undetected becausemost transgenic mouse experiments use hemizygousanimals. Unless the gene harboring the integrant dis-plays haploinsufficiency, no overt phenotype will beobserved. In contrast, production of homozygousmice may reveal a novel phenotype due to disruptionof both endogenous alleles where integration ofthe transgene has essentially produced an accidentalknockout mouse. Homozygotes are generated byintercrossing hemizygous animals within the sameline. Homozygous animals provide a practical advan-tage because all progeny will be transgenic, increasingthe number of mice available for experiments. If thetransgene integrates into a gene essential for viability,


no homozygotes will be obtained. Alternatively, asubtle change in the phenotype may occur in homo-zygotes. If so, it is critical to discern whether themodified phenotype is due to an increase in transgenecopy number (twofold increase in homozygotesover hemizygotes) or disruption of another gene thatimpinges on the phenotype.

While development of an unrelated phenotype isnot the initial goal of most transgenic mouse experi-ments, insertional mutagenesis can reveal novel genefunctions. In 2004, only about 10% of all mouse geneshad been knocked out (Austin et al., 2004). Thus,insertional mutagenesis represents an alternativemechanism for discovering the functions of previ-ously uncharacterized genes. In some cases, the unex-pected phenotype may mimic a human disease,providing a new genetic model for assessing underly-ing mechanisms. A further advantage of insertionalmutagenesis is the ability to use the transgene as amolecular tag to rapidly identify the disrupted gene.Transgene-mediated gene disruptionwas first discov-ered in a study where homozyogous mice developedlimb deformities. Positional cloning of the transgeneinsertion site ultimately led to the identification offormin genes (Woychik et al., 1985; Maas et al., 1990).

85.2.2 Transgene Composition

The precise composition of a transgene is dictatedby the question being asked, yet all transgenes,independent of purpose, are comprised of three prin-cipal elements. These include a promoter to directexpression to specific cells and tissues of interest,an expression cassette that encodes the RNA to beexpressed, and a eukaryotic polyadenylation sequence(Figure 1(a)). The promoter is typically from a differ-ent gene than that being expressed while the polyA+

sequence may or may not correspond to this gene.Many cDNAs lack polyA+ sequences due to the trun-cation of the 30 flanking region; thus, a heterologousconsensus sequence is engineered onto the end of thetransgene. Although it is not necessary to use a pro-moter or polyA+ sequence that is distinct from theexpression cassette, it is important to recognize thatuse of an uncharacterized promoter may result in lowexpression or activity in an inappropriate cell type.Hence, a select subset of promoters is routinely usedfor targeting expression to a particular tissue. Forexample, transgenes designed for mammary glandexpression usually contain the mouse mammarytumor virus (MMTV) or whey acidic protein (WAP)promoters (Yarus et al., 1996). These promoters have

been used for a large number of transgenes; thus, theirability to drive expression in the mammary gland isvirtually guaranteed. This is less of an issue with thepolyA+ sequence.

Extensive characterization of promoters that tar-get expression in the nervous system would greatlyexpand the repertoire of transgenic models to studydevelopment and behavior. This has lagged behindother organ systems such as the mammary glandor liver due to the diverse array of specialized celltypes and brain regions that require precise temporaland spatial targeting. Furthermore, neural functionextends beyond cell type and depends on connectivityand peripheral inputs, increasing the difficulty foridentifying promoter regulatory regions that can targetselected cells (Gaveriaux-Ruff andKieffer, 2007). Someof the more commonly used promoters/regulatoryelements include those from the genes encoding nestin(neural progenitor cells (Zimmerman et al., 1994)),tyrosine hydroxylase (dopaminergic (Sawamoto et al.,2001) and catecholaminergic (Savitt et al., 2005) neu-rons), and calcium-camodulin-dependent kinase IIa(CaMKIIa; forebrain (Mayford et al., 1996)). Signifi-cant advancements are being made in identifyinguseful regulatory regions for specified expressionof transgenes by the Gene Expression NervousSystem Atlas (GENSAT). This project is aimed atgenerating gene-expression maps in transgenic mice.It uses BAC-based transgenes to target expressionof enhanced green fluorescent protein (EGFP) tospecific regions of the brain and spinal cord. Utiliza-tion of BAC clones of a specific gene facilitates cap-turing sufficient regulatory sequences to recapitulateexpression of the endogenous gene.Mice produced bythis project are readily available and are useful fortracking individual cells in neuronal mapping stu-dies as well as electrophysiological recordings. Moreimportantly, expression patterns of individual BACsare readily observable within the searchable atlason the GENSAT website. These BACs can then beobtained and used as a vehicle to force expression of adifferent transgene to the same anatomical location.

In addition to selecting a highly active promoterregulatory region, it is often advantageous to usea genomic clone rather than a cDNAwithin the expres-sion cassette because introns increase transgene expres-sion (Brinster et al., 1988; Whitelaw et al., 1991; Choiet al., 1991; Palmiter et al., 1991). If a cDNA is utilized,an intron should be engineered into the transgene. Thiscan be from a heterologous gene or the same gene asthe cDNA. The inclusion of an intron likely enhancesseveral aspects of mRNA metabolism, including


increased transcription initiation and processivity,export through nuclear pores, and increased translationefficiency. The latter two processes are facilitatedby the formation of an exon junction complex (EJC)near the boundaries of exon–exon linkages. The posi-tion of the EJC is critical for the cell to recognize thematurity of mRNA, leading to nonsense-mediateddecay (NMD) if it occurs in the wrong position(Le Hir et al., 2003). Thus, in addition to includingan intron in the transgene cassette, its position withinthe cassette is also important, with placement at the 50

end being more effective than at the 30 end (Palmiteret al., 1991; Matsumoto et al., 1998).

Once the appropriate promoter, polyadenylationsequence, and intron are selected and the transgene iscloned into a vector, it should be removed from theplasmid prior to insertion into the mouse genome.Prokaryotic DNA can inhibit the expression level ofthe transgene without affecting its tissue-specific pat-tern (Townes et al., 1985; Palmiter and Brinster, 1986;Clark et al., 1997; Chevalier-Mariette et al., 2003).This may be due to the presence of CpG islandswhich are more densely packed in bacterial thaneukaryotic DNA (Chevalier-Mariette et al., 2003).Such islands are susceptible to hypermethylationand epigenetic silencing.

85.2.2.1 Reporter genes

One of the first applications of transgenic mouse tech-nology involved identifying promoter regulatoryregions of selected genes, extending conclusionsgleaned from in vitro promoter assays in immortalizedor transformed cell lines to a physiological context(Swift et al., 1984; Ornitz et al., 1985; Townes et al.,1985; Overbeek et al., 1985). Experimental outcomesfrom both approaches were often in agreement, sug-gesting that the in vitromodelswere effective analyticaltools. However, there were also many instances wherepromoter regulatory sequences identified in vitro wereinsufficient to convey appropriate expression in mice(Ross et al., 1990; Graves et al., 1991; Ray et al., 1995;Bennani-Baiti et al., 1998; Forrester et al., 1999). Thesediscrepancies have often been attributed to the absenceof chromatin regulatory elements within the transgene.However, it is also plausible that cell lines, whichtypically correspond to a single developmental state,are incapable of recapitulating the elaborate processesin the developing mouse that cause stepwise activationand repression of genes. Regardless of the mechanisticintricacies involved in promoter activation in trans-genic mice, this approach also spawned an importanttechnological advance. Specifically, robust promoters

were identified that were highly effective at reproduc-ibly directing expression of any protein of choice tospecific tissues and cell types. Transgenic mice alsoprovided tractable model systems for the study ofpromoter function where in vitro approaches werenonexistent; for example, this technology propelledidentification of regulatory mechanisms for geneswhose expressionwas confined to cells such as neurons(Dancinger et al., 1989; Forss-Petter et al., 1990) andpituitary gonadotropes (Bokar et al., 1989; Keri et al.,1994).

Promoter analyses, whether using cell culture ortransgenic mice, rely on the fundamental principlethat the expression level of the coding sequencedownstream of the promoter is directly related topromoter activity. In other words, the protein pro-duced by the transgene is subject to no other regu-latory mechanisms other than transcription. Thesecoding sequences are called reporter genes becausethey report on promoter activity. Two additionalproperties of reporter genes are that they are benign,that is, they do not alter the cell’s physiology inany way, and they are not intrinsic to a cell. Thelast criterion generates low background noise,making reporter assays highly sensitive. Reportergenes can encode a bacterial or viral enzyme, suchas b-galactosidase (lacZ), Herpes Simplex Virus thy-midine kinase (HSV-TK), or chloramphenicol acetyltransferase (CAT), or a protein from nonmammalianeukaryotes such as green fluorescent protein (GFP)or firefly luciferase. For all of these reporters, proteinexpression or enzymatic activity is readily assessedand has no impact on the physiology of mammaliancells. In some cases, a biologically active protein, suchas bovine growth hormone (bGH), has been usedbecause it can be readily measured in mouse serumand its overexpression alters growth and glucosehomeostasis (Palmiter et al., 1982, 1983). While itsatisfies some of the criteria of a reporter gene, miceexpressing bGH have a wide range of pathologies,making them unsuitable for promoter analyses.These animals were, however, useful for identifyingthe detrimental effects of excessive growth-hormoneproduction (Bartke et al., 1999).

Selection of a reporter gene depends on the ulti-mate objectives of the study. If identifying individualcells capable of activating a promoter is the goal, itwould be facilitated by a visually detectable reporterlike lacZ that produces a blue precipitate in cellsprovided with a chromogenic substrate (e.g., X-gal).LacZ is a useful marker for imaging cells in frozentissue sections. It can also be used to visualize live


cells, although this method is not ideal due to issuesrelated to substrate availability and harmful reac-tion conditions. In contrast, cells expressing GFPare readily detected in living cells with a fluorescencemicroscope without adding an exogenous substrate.GFP imaging can also be coupled with immunohis-tochemistry for other proteins (Takada et al., 1997;Hadjantonakis et al., 2003) and it has greater sensitiv-ity than lacZ (Chiocchetti et al., 1997). While instru-mental for evaluating expression in individual cells,neither GFP nor lacZ is easily quantified. Reportersthat produce measurable enzymatic activities in tis-sue lysates such as luciferase are more effective forcomparing the relative importance of selected regu-latory elements in a promoter.

In addition to indicating the relative promoteractivity, reporter genes can be used to identify andtrace specific cell types. Use of spectral variants ofGFP, such as YFP (yellow fluorescent protein) or RFP(red fluorescent protein), has enabled ex vivo imaging,where the impact of specific agents or drugs on indi-vidual cell behaviors can be evaluated in real time. Forexample, expression of YFP using the Thy 1 promoterlabels neurons in the preoptic area of the anteriorhypothalamus (POA-AH) (Feng et al., 2000), a regionthat is sexually dimorphic and controls various sexualbehaviors. Analysis of cell migration in organotypicslices from this region using live cell microscopymigration of cells within the POA-AH during embry-onic development is differentially regulated by estra-diol and dihydrotestosterone (Knoll et al., 2007). Thesestudies, enabled by the combined use of transgenicmice and cellular imaging, suggest that sexually dimor-phic behaviors may be determined, in part, by migra-tion patterns within the POA.

85.2.2.2 Indicator strains of mice

Imageable reporters can also be used to produceindicator mice, which are designed to convey quanti-fiable information on the in vivo activities of selectedproteins. Indicator mice are primarily generated toreport on the activity of a specific transcription factor.The transgenes usually contain a reporter gene that isunder the control of a minimal promoter linked tomultimerized consensus DNA-binding sites for thetranscription factor of interest. When the factor acti-vates endogenous genes, it will also induce expressionof the transgene reporter, which is biologically inert.Indicator mice are useful for determining when aspecific factor is activated; however, they are limitedto generating correlative data. This contrasts with theuse of overexpress ion or knockout (see Section 85.3)

models where a behavioral response can be measured.This second approach is also limited due to thepossibility that the overexpression/knockout modelmay have undergone a pathological change in thebrain. If so, it is difficult to discern whether a behav-ioral response is due directly to a change in transcrip-tion factor activity, or a consequence of pathology.The specific limitations of each approach can bediminished by designing studies that couple theuse of indicator mice with approaches that modifyprotein activity.

Indicator mice have been developed that measurethe activation status of several transcription factors inthe brain including Notch (Basak and Taylor, 2007);nuclear factor kappa B (NFkB; Lernbecher et al.,1993; Schmidt-Ullrich et al., 1996), estrogen receptor(Nagel et al., 2001; Ciana et al., 2003), AR (Ye et al.,2005), retinoic acid receptors (Balkan et al., 1992),b-catenin (Maretto et al., 2003), Smads 2/3 (Linet al., 2005), and others. One of the most informativeindicator mouse models reports the activation statusof CREB (Impey et al., 1998; Boer et al., 2007a), acAMP-responsive transcription factor whose activityis induced following phosphorylation by proteinkinase A (PKA). CREB-indicator mice thus reportnot only on activity of CREB, but also on fluxes incAMP and PKA activity. The transgene is comprisedof a lacZ reporter under the control of a minimalpromoter and multiple CREB-binding sites (cAMP-response elements or CRE). Use of these micerevealed that CREB activity is induced and main-tained in the hippocampus when an associative mem-ory is formed (Impey et al., 1998; Tully, 1998).A functional role for the CREB family of transcriptionfactors in hippocampus-dependent learning was laterconfirmed using a transgene to express a dominantnegative form of the protein in the forebrain (Pittengeret al., 2002). A different CREB-indicator mouse thatexpresses luciferase under the control of multimer-ized CREs has been useful for identifying addi-tional CREB-activating events in the brain. In thesemice, exposure to chronic psychosocial stress inducesCREB activity in the cerebellum, pons, colliculi, andhippocampus. Chronic treatment with the antide-pressant, imipramine, reversed this increase (Boeret al., 2007b), indicating that changes in CREB-mediated transcription occur during neuronal adap-tations associated with psychosocial stress.

A second type of indicator mouse uses biosensorproteins to report on changes in calcium flux, whichcan be used as a proxy for alterations in neuronalactivity. Upon binding to free calcium, fluorescent


calcium indicator proteins (FCIPs) undergo confor-mational changes that alter their fluorescence emis-sion spectra (Hasan et al., 2004). There are two typesof FCIPs. Cameleons use two spectral variants ofGFP linked to a calcium-sensing protein. When theprotein binds to calcium, a conformation change gen-erates a measurable shift in the fluorescence spec-trum (fluorescence resonance energy transfer orFRET; Miyawaki et al., 1997, 1999). The secondtype uses a calcium-sensing linker embedded withinGFP (Baird et al., 1999). This linker disrupts the 3-Dstructure of GFP attenuating its fluorescence. Uponbinding of calcium, the correct domain configurationoccurs, restoring fluorescence. FCIPs can be geneti-cally delivered to specific regions of the brain usingpromoter-directed transgene expression (Knopferet al., 2006). This permits increased spatial and tem-poral recording of brain activity compared to othernoninvasive imaging modalities such as functionalmagnetic resonance imaging. In addition, transgenesoffer increased selectivity of neuronal populationsover bulk loading of membrane-permeant calciumindicator dyes. Recent modification of the cameleonapproach has resulted in the production of transgenicmice with sustained sensor activity and a largedynamic range for detecting calcium oscillations inintact brains (Heim et al., 2007). The most intriguingaspect of this approach is the ability to detect changesin calcium flux in the entire cytosol of individualneurons, including secondary and tertiary dendrites(Heim et al., 2007).

FCIPs are particularly useful for measuring cal-cium oscillations in neurons that are scatteredthroughout the brain. For example, gonadotropin-releasing hormone (GnRH) neurons are diffuse inthe basal forebrain (Witkin et al., 1982), making loadedcalcium indicator dyes uninformative. The GnRHpromoter, which is highly specific for conveyingexpression of transgenes to GnRH neurons (Mellonet al., 1990), was used to direct restricted expression ofan FCIP to these neurons. This resulted in a modelwhere activity of GnRH neurons could be readilymeasuredwithin forebrain explants (Jasoni et al., 2007).

A third type of indicator mouse utilizes a reportergene that is inserted into (knocked-in) a specific geneusing homologous recombin ation (see Section 85. 3.2).Activity of the reporter gene is a direct measure ofthe expression of the gene into which it is inserted.In this type of mouse, the reporter gene is inserteddownstream of the promoter of the endogenous genebeing studied, usually in an exon. The reporter genewill be expressed in the same spatial and temporal

pattern as the endogenous gene. This paradigm hasbeen used to identify novel regions of sexual dimor-phism in the murine brain. Only a few anatomicallydistinct sexually dimorphic nuclei exist in the mousebrain, suggesting that gender-based differences inbehavior may extend beyond these nuclei. To testthis hypothesis, a novel reporter gene system wasconstructed that involved insertion of two reportersinto the endogenous AR gene. One of these reporterslabeled the nuclei of cells capable of expressingAR (nuclear-targeted lacZ), while the other labeledneuronal processes of the same cells with placentalalkaline phosphatase or PLAP (Shah et al., 2004).Hence, the nuclei and projections of sexually dimor-phic AR-expressing cells could be identified. Thisapproach revealed that there are many regions ofthe brain that express AR in a sexually dimorphicmanner. Some of these correspond to regions whoseneuronal activity (as demonstrated by Fos expression)changes with male mating, indicating that they maycontribute to gender-specific behaviors (Shah et al.,2004). These mice have also been used to discern AR-expressing neurons in the suprachiasmatic nucleus.Further studies revealed that these cells respond tophotic stimuli, leading to the hypothesis that theymay be important in androgen regulation of circadianbehaviors (Karatsoreos et al., 2007).

85.2.2.3 In vivo imaging

Genetically encoded imageable reporters haverecently transitioned into the realm of noninvasivein vivo imaging, where dynamic changes in biologicalactivity can be readily visualized in living animals.This has been facilitated by the development ofhighly sensitive optical imaging devices that detectchanges in bioluminescence or fluorescence. Lucifer-ase activity can be detected and quantified with acharge-coupled device (CCD) camera designed forhigh-efficiency photon detection after transgenicanimals are given an intraperitoneal or intravenousinjection of luciferin, a fluorogenic luciferase sub-strate (Contag and Bachmann, 2002). Distribution ofluciferin in the body is extensive; traversing theblood–brain and placental barriers, and the lightproduced upon luciferin cleavage is relatively resis-tant to scattering and tissue absorption. This tech-nique has been extensively utilized in cancer biologyto evaluate metastatic progression and therapeuticresponse (Gross and Piwinica-Worms, 2005). Its usehas been limited, however, in neurobiological researchdue to its intrinsically low spatial resolution. If cellularresolution is not required, this technique can be highly


informative. For example, a Smad-luciferase indicatormouse demonstrated that focal neocortical injury orexposure to endotoxin rapidly induces Smad2/3 sig-naling in the brain (Lin et al., 2005).

A CCD camera can also be used to detect fluores-cence activity generated by GFP, but low spatialresolution is still a concern. In contrast, multiphoton,confocal microscopy can be used to detect fluores-cence activity with subcellular resolution. Chronicimaging of transgenic mice can be performed, lead-ing to construction of a dynamic view of neuronalstructure and behavior. This approach involves theremoval of a small region of the skull and placementof an imaging window. The mice can then be repeat-edly imaged over several weeks during which theyare subjected to diverse exogenous stimuli. Thisimaging technique was used with transgenic miceexpressing enhanced-GFP in a subset of pyramidalneurons in the barrel cortex to show that dendriticspines, and hence synapses, undergo increasedturnover with sensory experience (Trachtenberget al., 2002). Similarly, monitoring cells expressingGFP in juxtaglomerular neurons of the olfactorybulb revealed that a subset of these neurons periodi-cally die and are replaced by new neurons in adults.These mice now provide an important model forstudying adult neurogenesis and turnover (Mizrahiet al., 2006).

In addition to neuroanatomical information,optical imaging tools can be used to appraise theactivities of individual proteins. A GFP-family mem-ber can be inserted into the coding region of anendogenous gene by homologous recombination(see Secti on 85.3.2), re sulting in the for mation of atagged, fusion protein. This protein will retain itsnormal pattern of expression and activity. Changesin the function and movement of the tagged proteincan then be monitored by confocal microscopy. Forexample, using eGFP to label the d-opioid receptor(DOR) permitted functional imaging of thisreceptor’s subcellular trafficking in living animals(Scherrer et al., 2006). By treating these mice withvarious drugs that promote endocytosis, behavioralpatterns could be compared with the level of receptorinternalization. These studies provided direct in vivoevidence that receptor redistribution correlates withchanges in locomotor activity.

There is no doubt that in vivo imaging willbecome an integral component of the neuroscientist’stoolkit where mechanisms that were first identifiedin vitro can now be evaluated within a living organ-ism. The ability to assess the functions of cells,

subcellular organelles, and even proteins in realtime provides considerable depth and physiologicalrelevance to studies that were once confined toisolated cell models.

85.2.2.4 Protein overexpression

Themost extensively used transgenic mouse paradigminvolves targeted overexpression of various wild-typeor mutant proteins in selected cells. Depending on thequestions being asked, these proteins can be wild-type,dominant negative, or constitutively active isoforms.In addition, a protein can be mis-expressed in anincorrect cell type. Overexpression models havebeen frequently employed to determine if a proteinhas oncogenic properties. Expression of oncogenesin transgenic mice also presents an opportunityto develop novel cell lines from resulting tumors.This approach was advantageous for developingthe hypothalamic GT1-7 neuronal cell line thatexpresses GnRH. The low abundance and scatteredappearance of GnRH neurons in the hypothalamusare significant barriers to studying their function.However, production of the GT1-7 cell line has sup-planted some of these limitations. These cells wereisolated from hypothalamic tumors arising in trans-genic mice expressing the simian virus 40 (SV40)T-antigen under control of the GnRH promoter(Mellon et al., 1990). Remarkably, they synchronouslysecreteGnRH in a pulsatile mannerwith an interpulseinterval similar to that observed in mice (Wetsel et al.,1992). This mouse tumorigenesis approach resulted inan essential means for identifying regulatory mechan-isms in GnRH-secreting neurons in vitro.

Mis-expression of a protein is particularly usefulfor determining if it controls cell fate. This approachwas useful for confirming that the Sry gene is thesex-determining factor in mammals. When a largegenomic fragment of the Y chromosome surroundingthe Sry gene was used to make transgenic mice,genetic females (XX) with this transgene were phe-notypically male. This included formation of testes(Koopman et al., 1991) and acquisition of some malebehaviors (De Vries et al., 2002). This outcome notonly demonstrated that the Sry gene was sufficient forgender determination; it also revealed that genesother than those on the Y chromosome contributeto male behavior.

Overexpression of proteins is also useful for dis-cerning the roles of proteins in pathological pro-cesses. This approach has been used to demonstratethat excessive amounts of either the glucocorticoid ormineralocorticoid receptors within the forebrain

TSP shRNA

AAAAA

siRNA

shRNA

Target mRNADegraded mRNA

Figure 3 Transgene-mediated gene silencing using an

shRNA-expressing construct. The shRNA is expressed

under the control of a tissue-specific promoter and isprocessed by intrinsic enzymes. The shRNA binds to the

targeted mRNA and induces its degradation.


alter anxiety and depression-like behaviors, but withopposite effects (Wei et al., 2004; Rozeboom et al.,2007). These studies indicate that the two steroidreceptors and their adrenal-derived ligands distinctlycontrol emotional reactivity. They also suggest thatreceptor variants may modulate susceptibility tomood disorders, opening new avenues for the discov-ery of genetic polymorphisms that correlate withanxiety and depression.

85.2.2.5 Transgene-mediated gene silencingSelectively blocking the function of a protein isoften more appropriate than over- or mis-expression.This can be accomplished through three routes: dis-rupting the protein-coding gene (knockout, seeSection 85. 3), expre ssing a dominant negative for mof the protein, or reducing the protein’s expressionby preventing its translation or the accumulation ofits mRNA. Gene disruption is the only approachwhere complete elimination of a protein can beassured. However, it requires a significant investmentof time and resources. If the experimental questiondoes not mandate complete eradication of the pro-tein, one of the other two approaches may be morepractical.

Engineering a dominant negative protein neces-sitates sufficient understanding of its structure–function relationships to endow it with the ability toselectively abrogate activity of its endogenous coun-terpart. The mutant must retain its ability to associatewith proteins within the cell, but prevent transmis-sion of a signal. In some cases, this could involvetargeting protein dimers. If both members of adimer must be functional to convey a signal, mutationof one subunit should inactivate dimers containingthe mutant. This is how most dominant negativeforms of nuclear receptors function. Alternatively,a dominant negative protein may act as a sinkthat efficiently sequesters effector proteins, but can-not transduce a signal. Many dominant negativesof small ras-related GTPases act in this fashion.A C-terminal truncation mutant of disrupted-in-schizophrenia-1 (DISC1) acts as a dominant negativeby interacting with the endogenous protein andinducing its mislocalization (Kamiya et al., 2005).Expression of the mutant in pyramidal neurons ofthe medial prefrontal cortex and in granule neuronsof the dentate gyrus causes asymmetric alterations inlateral ventricle size, interneuron deficits, and hyper-activity (Hikida et al., 2007). These changes are similarto those observed in schizophrenic patients suggestingthat DISC1 may be an important contributor to this

disease and that these mice may be useful models fortesting this possibility.

Another method for reducing protein activityutilizes RNA-mediated, gene-specific silencing orRNA interference (RNAi), the characterization ofwhich garnered the Nobel prize for Andrew Fireand Craig Mello in 2006. There are several RNAitechniques and all reduce production of a targetmRNA’s cognate protein. Depending on the methodand transgene expression levels, the efficiency ofknockdown can range from modest to nearly com-plete elimination. RNAi takes advantage of endoge-nous cellular machinery that processes microRNAs(miRNAs) and defends against viral infections. Shortoligoribonucleotides are produced that sequence-specifically hybridize to endogenous mRNAs. Thehost cell recognizes this RNA duplex and, dependingon its extent of complementarity with the targetmRNA, either induces degradation or inhibits itstranslation (Leung and Whittaker, 2005; Martin andCaplan, 2007). Two approaches are used to employRNAi in a transgenic mouse and both result in thesynthesis of a short, interfering RNA (siRNA) duplex.A well-characterized promoter that targets the cellsof interest is used to convey expression of eithera short hairpin RNA (shRNA; Figure 3) or anmiRNA backbone, both of which are processedinto an siRNA duplex that has complementarityto the target mRNA. There are several web-basedalgorithms available to facilitate generation of appro-priate duplexes. Although both shRNA and miRNA


have been used in transgenic mice, miRNA has twoadvantages. First, miRNA appears to silence endoge-nous mRNAs more efficiently than shRNAs (Changet al., 2006). Second, miRNAs are produced as largerRNA transcripts that can be quantified by Northernblotting or RT-PCR, whereas shRNAs are too shortfor these methods.

In addition to being more rapid, there are severaladvantages of using RNAi transgenics comparedto conventional knockouts. Unlike targeted genedisruption, mice with silencing transgenes do notrequire breeding to homozygosity to observe pheno-typic effects because the transgene acts as a dominantallele. RNAi transgenes are also relatively simple toconstruct compared to the complex targeting vectorsnecessary for knockouts. In addition, an RNAi trans-gene will be subject to position variegation, generat-ing variable degrees of silencing across individuallines of mice. These lines can be viewed as harboringhypomorphic alleles (Peng et al., 2006) and can beused to assess dose–response relationships for theprotein of interest. In contrast, knockouts yieldall-or-none levels of protein expression.

There are also several limitations of using RNAi-mediated gene silencing. First, complete eliminationof the targeted protein is difficult to accomplish andcannot be assured. If an RNAi transgene does not yielda phenotype it is possible that low-level expression ofthe protein is sufficient. The only way to exclude itsrole is by knocking out the gene. An extension of thislimitation is the dependency on promoter regulatoryregions to convey shRNA/miRNA expression. Thepromoter must be sufficiently robust to produceenough siRNA to silence gene expression. A secondmajor drawback is the potential for off-target effectsof the siRNA. The duplexes are short and rules gov-erning hybridization with target mRNAs are notcompletely known; hence, they may target otherunknown mRNAs. This off-target silencing mayunderlie the phenotypic changes. This is readilyaddressed by constructing multiple lines of micewith RNAi transgenes that bind to different regionsof the target mRNA. A consistent phenotype amongthese lines suggests that the targeted protein does,indeed, play a role.

RNAi has provided a major advance in the abilityto generate rats with reduced expression of a targetprotein that overcomes the technical challenges ofproducing a knockout in this species. Lentiviral vec-tors that express shRNA can be delivered to ratembryos following injection of viral particles under

the zona pellucida of fertilized oocytes (Dann, 2007).This requires very high titers of virus, but counter-acts the technical difficulties associated with pronu-clear injection in rats. The dominant activity ofshRNAs makes the founder animals (i.e., thoseinfected as embryos) useful for analyzing phenotypicchanges, although the possibility and impact of trans-gene mosaicism must be recognized. If the transgeneis integrated into the germ line, heritable silencingcan also be observed through multiple generations.This approach was recently employed to assess thefunction of deleted in azoospermia-like (DAZL) indevelopment of rat male germ cells (Dann et al.,2006). The DAZL-targeted shRNA induced com-plete male infertility in the presence of normal mat-ing behavior. The rats were unable to produce maturesperm and underwent progressive loss of germ cells,consistent with what was observed in DAZL knock-out mice (Ruggiu et al., 1997). Thus, this techniqueappears to be a viable approach for studying genefunction in rats and may provide a new mechanismfor evaluating the genetic basis of behavior in thiswell-established model.

85.2.2.6 Cell-specific ablation and

inactivationAblation of sets of cells has been an important tool forexamining lineage relationships as well as developingmodels of neuronal loss. Before the advent of trans-genic animals, this was accomplished using surgicaltechniques or cell-selective toxins. While informa-tive, unveiling the contributions of particular cellpopulations in development and disease can be facili-tated by precisely eliminating individual cell types inselected regions of the brain. Transgene-mediatedablation of cells affords this selectivity via geneticallytargeting expression of toxic gene products (toxi-genes) to individual cell types. Two toxins havebeen used extensively: HSV-TK and diphtheriatoxin A chain (DT-A). Due to limitations associatedwith HSV-TK (i.e., transgene silencing, low potency,and toxicity in male spermatids (Palmiter, 2001)), wewill confine this discussion to diphtheria toxin. Thistoxin is comprised of two protein subunits: A andB chain. The B chain binds to HB-EGF, a receptornecessary for endocytosis of diphtheria toxin. TheA-chain ADP ribosylates elongation factor 2 (EF2),thereby preventing protein synthesis and inducingcell death (Palmiter et al., 1987). The DT-A chaincan be expressed from a transgene in selected cells.


It is highly toxic and expression of just one DT-Amolecule is sufficient to induce cell death (Palmiter,2001). Most importantly, this effect is cell autono-mous: DT-A will only kill cells in which it isexpressed. It cannot enter adjacent cells becauseDT-B is absent. By judiciously selecting a promoterwith the desired temporal and spatial pattern ofexpression, a specific set of cells can be eliminatedwith precision. The consequences of that cell loss canthen be appraised in the embryo or an adult.

The considerable toxicity of DT-A can be problem-atic. If a promoter is leaky, meaning that it is modestlyexpressed in the wrong cells or at an inappropriatetime, those cells will also die and potentially contributeto the phenotype. Alternatively, a promoter may beactive during embryonic development, but it may bemore informative to ablate cells after that time. Leakyexpression of DT-A can be so severe that it inducesembryonic lethality. To address these issues, an induc-ible approach has been developed that takes advan-tage of the differing affinities of the diphtheria toxinreceptor (DTR, also known as HB-EGF) betweenmice and man. Mouse DTR has a very low affinityfor DT-B; hence, mice are insensitive to diphtheriatoxin. Sensitivity can be induced using transgene-mediated expression of the human isoform of DTR,which binds DT-B with 105-fold higher affinity (Saitoet al., 2001). Using an appropriate promoter, expressionof human DTR can be targeted to selected cells,making them uniquely sensitive to an injection ofpurified diphtheria toxin. This method offers theadvantage of selective inducibility for a highly toxicprotein. It has been used to address the importance ofAgouti-related peptide (AgRP)-expressing neurons inmodulating feeding behavior. AgRP neurons are tar-gets of several energy balance regulating hormonessuch as leptin, insulin, and ghrelin. However, geneticdisruption of their two primary neuropeptides (neuro-peptide Y and AgRP) fails to alter feeding behavior(Qian et al., 2002), suggesting that these cells may notregulate feeding. This supposition was challengedwhen selective ablation of AgRP neurons induced theimmediate onset of anorexia (Gropp et al., 2005).

Another cell-specific ablation method has beendeveloped that is unique for neurons. It involvestargeted expression of the C-terminal portion ofthe human ataxin-3 protein from a patient withMachado–Joseph disease (Bewick et al., 2005). Thetransgenic protein contains 77 polyglutamine repeats,forms nuclear aggregates, and subsequently inducescell death. Expression in AgRP neurons resulted in a

similar, although milder, hypophagic phenotype asobserved with the DTR/diphtheria-toxin paradigm(Bewick et al., 2005). The reduced severity of thephenotype is likely due to differences in the relativetoxicities of the two transgenic proteins. Together,these studies revealed that AgRP-expressing neuronsmust produce additional neuromodulators that con-trol feeding. These studies demonstrate that cell-specific ablation in adults can be highly effective fordiscerning the potential role of classes of neurons inregulating various aspects of hormonally controlledbehavior, such as feeding.

Ablation of cells with toxigenes reveals their rela-tive importance in neuronal specification and/orintercellular communication. Further refinement ofthis method to ablate synaptic transmission ratherthan entire cells would generate further insightsinto the role of intercellular communications in thecontrol of behavior. A technique has recently beendeveloped that permits selective, inducible, andreversible silencing of synaptic transmission from aspecific population of targeted neurons. This hasbeen accomplished using a combination of a geneti-cally engineered fusion protein (molecules forinactivation of synaptic transmission or MISTs) andtreatment with an agent that inactivates that protein.MISTs are chimeric proteins comprised of a portionof a synaptic vesicle protein linked to a smallmolecule-binding domain of another protein suchas FK506-binding protein (FKBP). The seconddomainundergoes inducible dimerization with the addition ofa drug (e.g., FK506). Exposure to the drug cross-linkstwo MIST proteins, disrupting their normal functionand inactivating synaptic vesicle cycling.This approachwas first used to modulate synaptic vesicle activity incerebellar Purkinje cells using the L7/pcp-2 (Purkinjecell protein-2) promoter to drive expression of aMISTlinked to VAMP/Syb (vesicle-associated membraneprotein/synaptobrevin) (Karpova et al., 2005). MIST-expressing mice were indistinguishable from theirwild-type counterparts until they were treated withthe dimerizer. Upon treatment, transgenic animalshad significant deficits in learning and performancein the rotarod test compared to their wild-typecounterparts, indicating that sustained input fromPurkinje cells is necessary for motor learning and per-formance in adults. This technique can now be used totemporally disrupt synaptic transmission from anycell type for which a selective promoter has beenidentified, opening new avenues for defining signalsinvolved in neuronal development and plasticity.

Target transgene (−Dox)

O O

Effector transgene

tTATSP

P EC A+

A+

D

DD D

Doxycycline

Target transgene (+Dox)

O O P EC A+

Figure 4 The tet-off system for regulating transgene

expression requires two transgenes: a target with anexpression cassette for the desired protein and an effector

that expresses the tTA protein. tTA will bind to the target

transgene and activate transcription. In the presence of


85.2.3 Inducible/Conditional Expression ofTransgenes

Naturally occurring promoters provide useful tools forconveying transgene expression to selected cell types.However, experimental questions often require that thetransgene be expressed within a restricted window oftime, such as during a specific stage of development.While this type of control may occasionally be con-veyed by a natural promoter, it is usually difficult toobtain the desired temporal precision. To address thisissue, several binary transgenic systems have beendeveloped that permit inducible activation/suppres-sion of a transgene. All of these systems utilize twotransgenes: (1) a target transgene that is capable ofexpressing the desired mRNA/protein upon activationand (2) an effector transgene that controls activityof thetarget (Lewandowski, 2001). Together, they provide agenetic switch that can be induced at the desired time.Depending on the particular system used, transgeneexpression may also be silenced during a specifiedwindow. The most common binary systems currentlyutilize ligand-regulated transcriptional control orsite-specific DNA recombination.

doxycycline, tTA falls off the DNA and transcription of thetarget is terminated.

85.2.3.1 Ligand-regulated transgeneexpression

Tight temporal control of transgene expression canbe achieved with a ligand-regulated system where thetarget transgene contains an expression cassette forthe desired gene product under control of a syntheticpromoter. The promoter is engineered to containcis-acting regulatory elements that bind a transcrip-tion factor whose activity is modulated by the pres-ence or absence of a particular ligand (Lewandowski,2001). The transcription factor is delivered to cells ofinterest using an effector transgene containing a pro-moter that directs expression of the factor with thedesired temporal and spatial pattern. The target andeffector transgenes are separately used to producetransgenic mice and neither should generate a phe-notype. In contrast, bi-transgenic animals generatedby intercrossing the target and effector lines may havea phenotype depending on whether the ligand shutsoff or turns on target transgene expression.

One of themost common ligand-regulated systemstakes advantage of the tetracycline repressor (tetR)derived from the tet operon of Escherichia coli(Figure 4). tetR binds to a 19-bp recognitionsequence (tetO) in DNA and multimers of this ele-ment can be engineered into the promoter regulatoryregion of target transgenes. In bacteria, tetR is a

transcriptional repressor responsible for sensingexposure to tetracycline. Upon binding to tetracy-cline, it undergoes a conformational change and dis-sociates from DNA, leading to activation of theoperon. A chimera of the tetR protein known as tTA(tet transactivator) has been developed for use ineukaryotic cells by adding the transcription activationdomain from the HSV VP16 protein. The tetR por-tion of the fusion protein bestows sequence-specificDNA binding and tetracycline responsiveness whilethe VP16 domain contributes a transcription activa-tion domain that is recognized by eukaryotictranscriptional machinery. It can be delivered tocells in the form of an effector transgene. Whenbound to regulatory elements within a target trans-gene, tTAwill induce transcription. In the presence oftetracycline, tTA is released and target transgeneactivity is attenuated. This system is known as tet-off because the transgene is silenced by tetracyclineanalogs. Although this system was developed foractivation by tetracycline, its analog, doxycycline ismore commonly used because it is capable of crossingthe blood–brain barrier. This property makes itparticularly advantageous for induction of transgeneexpression in brain research (Chen et al., 1998).

Dormant transgene

ECUbP lacZ

CreTSP

Effector transgene

EC

UbP

A+

A+

A+

A+A+

ECUbP

Activated transgene

Cre-mediatedrecombination

lacZ

lacZA

+

A

+

C

C CCC

CC

CC

C

Figure 5 Recombinase-mediated transgene activation.

The dormant transgene uses a ubiquitously active promoter(UbP) to express b-galactosidase (lacZ) in all tissues. The

expression cassette (EC) downstream of lacZ is not

expressed and the lacZ cassette is flanked by loxP

sequences (black triangles) to permit its later removal. Theeffector transgene expresses Cre recombinase (C) under

the control of a tissue-specific promoter (TSP). In any cell

that expresses Cre, the lacZ cassette is excised from the

dormant transgene, leaving only a residual loxP motif andactivating the expression cassette.


There are two major limitations of the tet-offapproach. First, target gene regulation can be highlyvariable, with leaky expression being observed in thepresence of doxycycline. Second, to block transgeneexpression, all animals must be continuously treatedwith doxycycline until a specific experiment is per-formed. This includes embryonic development.Although doxycycline is relatively nontoxic, chronictreatment is labor intensive and there is some evi-dence that it may adversely affect placental and fetaldevelopment (Moutier et al., 2003). To address theseshortcomings, mutations have been made in tTA thatreverse its DNA-binding properties, inducing bindingwhen tetracycline analogs are present and causingrelease in their absence (Gossen et al., 1995). Thisreverse tetracycline activator (rtTA) provides a tet-onsystem with doxycycline-activating gene expression.With this approach, mice only require treatment duringthe desired time frame for target transgene expression.

Many other ligand-inducible systems exist. Mostof these also use a binary approach and each has itsown strengths and limitations. Together, these toolsprovide precise temporal control of transgene expres-sion. Many are also amenable to ligand titration,providing dose–response relationships for the over-expressed protein. Ligand-regulated systems can alsobe used for add-back experiments in knockout ani-mals. This permits selective restoration of a particu-lar protein in a temporally and spatially controlledmanner. For example, targeted disruption of PKCeincreases alcohol sensitivity and decreases con-sumption, which could be due to pleitropic effectsresulting from global loss of this protein. A doxycy-cline-regulated transgene made it feasible to restorePKCe expression only in the brain (Choi et al., 2002).This reversed the alcohol-sensitivity phenotype,revealing a brain-specific role for PKCe in regulatingalcohol consumption.

85.2.3.2 Recombination-induced transgene

expression

Another binary system used to induce transgeneexpression employs a recombination event as themolecular switch (Figure 5). This approach requiresa dormant transgene that is inactive until a recombi-nation event has occurred. That event is executed bya nonmammalian DNA recombinase encoded by asecond transgene. The two recombinases used thusfar are Cre and FLP, derived from bacteriophage P1and Saccharomyces cerevisiae, respectively. Both recog-nize pairs of 34-bp consensus sequences (termed loxPfor Cre and FRT for FLP) within DNA. Depending

on the orientation of the paired sequences, theseenzymes will invert, excise, duplicate, or translocatethe stretch of nucleotides between the recognitionmotifs. Mere expression in mammalian cells is suffi-cient for their activity because neither requires acces-sory proteins. Using a transgenic approach, therecombinases can be expressed in the cells of interestby selecting an appropriate promoter. In most cases,recombinase expression has minimal effects on thehost genome and mice with the recombinase trans-gene should have no detectable phenotype. There areseveral Cre recombinase-expressing transgenics cur-rently available that target the nervous system(Gaveriaux-Ruff and Kieffer, 2007), and many canbe purchased from The Jackson Laboratory. When-ever a new Cre mouse is developed, it must bevalidated to avoid using animals where Cre isexpressed at low levels or in an undesired tissue.This can be accomplished by breeding the novel


recombinase mouse with a ROSA26 Cre reporteranimal (Soriano, 1999), which will result in lacZexpression in all cells that express the recombinase.

To activate gene expression with a recombinationevent, recombinase transgenics are bred with miceharboring a dormant transgene that usually has aninhibitory sequence engineered between the tworecombinase recognition motifs. When the recombi-nase removes this intervening sequence, the dormanttransgene is activated. One of the most straightfor-ward approaches uses a transgene with a strong tran-scriptional or translational stop sequence precedingthe expression cassette. This sequence is flanked byrecognition sequences for the recombinase. For loxPsites, this is known as a ‘floxed’ sequence for flanked byloxP. The protein encoded by the expression cassettewill be produced only after the inhibitory sequence isremoved by the recombinase. Without the recombi-nase, animals harboring just the dormant transgene arephenotypically normal. In contrast, bi-transgenic micewill undergo recombination, permanently remove thestop sequence and activate the dormant transgene.Depending on how mating pairs are established, pro-duction of bi-transgenic animals can also generatesingle transgenics. These mice provide ideal controlsbecause they should be phenotypically normal eventhough they contain one transgene or the other.

The irreversible genetic change provided by arecombinase can be used for cell-fate mapping if animageable reporter is placed downstream of the stopin the dormant transgene. The reporter can only bedetected after a recombination event. If recombinaseexpression is limited to precursor/stem cells using anappropriate promoter, only the progeny of those cellswill be visible. Hence, promoters from genes thatencode markers of precursor cells are highly usefulfor lineage tracking. For example, using the glialfibrillary acidic protein (GFAP) promoter to conveyCre expression has revealed that astroglia present inpostnatal day 5 pups are precursors of multiple celltypes including neurons, astrocytes, and oligoden-drocytes (Ganat et al., 2006).

In addition to fate mapping, this approach can beused to trace individual neurons, if the imageablereporter protein is in the cytoplasm. A recent andexciting extension of this technique resulted in theBrainbow system (Livet et al., 2007), which inducesrecombination of combinatorial transgenes that con-tain numerous spectral variants of GFP. These var-iants are expressed in different ratios depending onstochastic Cre-induced recombination events. The

numerous combinations of fluorescent proteins canyield up to 90 discernable colors, permitting uniquelabeling of adjacent cells, their processes, and synap-tic contacts within a section of the brain. This modelwill undoubtedly be useful for producing a detailedmap of differences between the neuronal circuitry ofnormal and diseased brains.

While Cre is generally considered nontoxic inmammalian cells, high expression can induce chro-mosomal damage due to recognition of cryptic rec-ognition sites in the genome (Schmidt et al., 2000;Loonstra et al., 2001). This situation can be avoidedby titrating Cre levels through judicious selection ofthe specific line of Cre expressing animals to be used.Alternatively, expression or activity of Cre can bedirectly regulated. Expression levels can be manipu-lated by using an inducible promoter upstream of theCre-coding sequence. This ultimately requires pro-duction of triple transgenic animals: (1) inducible Cretransgene, (2) transgene encoding an inducible tran-scriptional regulator, and (3) the Cre target trans-gene). Alternatively, Cre activity can be controlledby using a version of the recombinase that is ligandinducible. Fusion of Cre to a mutated ligand-bindingdomain (LBD) of the estrogen receptor (ERT2) causesits sequestration in the cytoplasm until it binds theligand 4 hydroxytamoxifen (OHT) (Brocard et al.,1997). Upon OHT binding, the CreERT2 proteintranslocates to the nucleus where it can induce site-specific recombination.

85.3 Homologous Recombination ofForeign DNA into the Murine Genome

Random insertion of DNA into the mouse genome(i.e., transgenics) provides numerous opportunitiesfor understanding the function of cells and proteinswithin a physiological/pathological context. How-ever, a major limitation of this technique is the inabil-ity to completely and verifiably eliminate proteins.As discussed above, even when using highly effectivesilencing transgenes, the possibility remains thatresidual expression is sufficient to obscure a pheno-type. It is also unknown whether RNAi transgenescan be used to efficiently reduce expression ofmiRNAs, which are involved in a plethora of regu-latory processes. In addition, transgenic approachesare unable to replace endogenous genes with geneticvariants. Consequently, the effects of a mutant pro-tein can only be studied in the context of endogenous


protein expression, which may or may not recapitu-late specific human genetic diseases.

In contrast to transgenic approaches, homologousrecombination can be used to insert foreign DNAinto preselected sites in the chromatin. This isknown as targeted gene disruption or production ofa knockout mouse. This tool was made possible by theadvent of two technologies: isolation of totipotentmouse ES cell lines that populate all cell lineagesand refinement of molecular tools that facilitateselection and identification of cells that have under-gone recombination. Combining these tools permit-ted targeted excision and replacement of regions ofthe genome with exceptional molecular precision,ultimately yielding technology that can be used tomodify sequences in the genome that range in sizefrom single nucleotide substitutions to the elimina-tion of entire genes.

85.3.1 General Principles

Homologous recombination can be used to disruptgenes or replace endogenous genes with geneticvariants. In all cases, the starting point is geneticmanipulation of ES cells. These cells can be grownex vivo and foreign DNA introduced into them bystandard transfection methods. They can also popu-late the murine germ line providing cloned animalswith heritable genetic change. Insertion of foreignDNA into a preselected locus in ES cells requireshomologous recombination. By virtue of comple-mentarity, exogenous DNA aligns with its counter-part in the genome and the cell’s machinery induces arecombination event, swapping a region of the endog-enous chromosome for the targeting DNA. Largestretches of matching sequences provide the nucleat-ing point for homologous recombination; however,they do not guarantee that this event will occur.Instead, foreign DNA integrates randomly into thegenome with relatively high frequency, while homo-logous recombination is an extremely rare event.This has required the development of various toolsto increase the odds of homologous recombinationas well as select ES cells that have undergone thisprocess.

By 2007, approximately 12 000 genes had beendisrupted by targeted mutagenesis, accounting fornearly half of the protein-coding genes in the mousegenome (Oliver et al., 2007). A concerted effort now isunderway to disrupt every protein-coding gene. TheKnockout Mouse Project (KOMP) is an NIH-funded

effort to provide a comprehensive public resource ofmouse ES cell lines that harbor null mutations inevery gene. The goal of KOMP is to provide knockoutmodels that have undergone standardized, stepwisephenotyping as well as increase availability of thesemodels (Austin et al., 2004).

Even though gene disruption technology hasbecome a cornerstone fromwhichmuch of our under-standing of physiological/pathological functions ofindividual genes has been built, the targeted inactiva-tion of a gene can sometimes generate unexpectedresults that conflict with established human patholo-gies. Two clear examples of discordance involve thep53 and Rb tumor suppressors (Classon and Harlow,2002; Johnson and Attardi, 2006). Although mice har-boring mutant alleles of these genes are cancer prone,the spectrum of tumors is distinct from human tumorsyndromes that arise from mutations in these genes.Such discrepancies can occur for multiple reasons, themost obvious of which is that the gene’s function maybe different inmice versus humans.When unexpectedphenotypes arise, it is important to characterize themouse model to determine if it has any biochemicalsimilarities to humans. If so, this would suggest thatthe protein has the same basic functions in both spe-cies, but the downstream responses to the mutationmay be species specific. Another underlying causeof discordant phenotypes can be the evaluation ofdifferent types of mutations in mice compared tohumans. Patients may have a point mutation ina gene whereas mouse models usually involve com-plete gene disruption. The point mutation may notcompletely inactivate the protein whereas targetedgene disruption does. This can be readily addressedby comparing humanized mouse models where themouse gene is replaced (knocked in) by either thewild-type human isoform or its mutant. Lastly, diver-gent phenotypes may be caused by species-specificdevelopmental responses. In knockouts, complete lossof a protein early during embryogenesis may induceirreversible developmental changes that would notoccur in a human with a more subtle mutation.The vast sequelae of molecular events that occurfollowing this loss may generate extensive pheno-typic changes or initiate compensatory mechanismsthat would not be at play in humans. This is parti-cularly important for studies examining behavioralchanges because a developmental alteration couldchange the fundamental biology of a specific regionof the brain. Behavioral phenotypes will undoubtedlyoccur in response to this developmental defect,


but attributing a behavior specifically to that generather than the global change in brain functionwould be difficult.

85.3.1.1 Targeting vectors and gene

disruption in ES cells

The first step in making a knockout mouse is gener-ating a targeting vector that will selectively recom-bine with an endogenous gene (Figure 6(a)). Thisvector has several key features that permit its inte-gration into a specific site in the genome and selec-tion for this relatively rare event. Targeting vectorsmust have long stretches of sequence that are identi-cal to the gene being targeted. These act as homingagents to the gene. The most common vector type is a

1

1

Targeting vector

Targeted gene

(a)

ES cells

targetingvector

Transfect

Recipient

ChimeraChimera

Breed(b)

Figure 6 (a) Standard targeting vector with positive/negative sembedded between two arms of homologous sequence that ar

nucleating sites for homologous recombination. The negative se

scheme for transfecting and selecting ES cells that have undergo

homologous recombination and are insensitive to gancyclovir. Cegancyclovir. These cells are injected into donor blastocysts to pr

founder heterozygotes can be obtained and a line of mice estab

replacement or O vector. Two regions of homology(the arms of theO) flank both sides of the sequence tobe inserted (the loop of the O). With this type ofvector, two recombination events occur; one oneach side of the foreign DNA. This promotes swap-ping of the foreign and endogenous sequences ratherthan simple insertion of the foreign DNA into thatlocus (Hasty et al., 1991b; Thomas et al., 1992). Largeregions of homology are necessary for aligning thevector sequence with the complementary genomicregion and providing a site for the cell’s endogenousmachinery to perform the recombination process.The optimal lengths of the homologous sequenceshave not yet been established, but rates of recombi-nation clearly increase with the size of the region of

32

neor

3 TK

neo

selection

Gancyclovirselection

Inject into

blastocysts

Founderheterozygote

Confirm

genotype

election markers. The positive selection marker (neor) ise identical to the gene to be knocked out. These arms are

lection marker (TK) is on one end of the vector. (b) Typical

ne homologous recombination. White cells have undergone

lls with random integration are speckled and are sensitive tooduce chimeric mice. If ES cells contributed to the germ line,

lished.


homology, up to about 10 Kb. The smallest regionthat can be used is about 500 bp (Hasty et al., 1991a;Deng and Capecchi, 1992). It is likely that the sizerequirement may also be locus dependent, withsome loci being highly resistant to this process,potentially due to chromatin configuration (Glaseret al., 2005). Publicly available BAC libraries thatspan the mouse genome have greatly facilitated theisolation of homologous sequences. A library clonecan now be identified through Internet-accessibledatabases and purchased from various genome repo-sitories. In addition to length, the genetic sourceof the homologous sequence is important. It shouldbe isogenic with the ES cell line. Only two types ofES cell lines are currently available, differing bythe mouse strain of origin: 129 and C57BL/6, with129 being the most frequently used. Fortunately,the BAC libraries described above include bothof these strains.

The homology arms are a vehicle for inserting aforeign sequence into a specified locus. This insertionaccomplishes two goals. First, it replaces essentialregions of the targeted gene (e.g., exons), causinginactivation. Second, it provides a mechanism forselecting cells that have integrated the foreign DNAinto their genome. For most genes, it is not possible todevise a rapid, efficient screen that will immediatelyidentify cells that have undergone an integration/inactivation event. Inclusion of a positive selectablemarker such as a neomycin (neor ) or hygromycin (hph)resistance gene in the targeting sequence providesprotection from antibiotics such as G418 that arenormally toxic to the cells. The resistance gene mayor may not have a promoter of its own. Including apromoter ensures that the selection marker will beexpressed whether it is integrated into a gene or not.However, if an exon is being targeted, it is moreprudent to engineer the vector in a way that theresistance gene will integrate in-frame in that exon.This increases the stringency of the screen becausethe resistance gene must be downstream of a pro-moter and within an exon to be expressed. Whenselecting an exon to disrupt, the 50-most exons aremost effective because the resistance gene alsoincludes a polyadenylation sequence that terminatestranscription of the remainder of the endogenousgene. This prevents unexpected synthesis of atruncated protein that may have residual activity orexpression of a full-length fusion protein that has aninserted resistance domain, but remains functional.

The strategies described above increase the fre-quency of homologous recombination and convey a

stringent selection scheme, yet the comparativelyhigh efficiency of random insertion still overridesthese tools. To eliminate cells that have undergonerandom integration of the foreign DNA, the targetingvector includes another essential selection compo-nent. A negative selectable marker gene is placed onthe outside end of one of the homology arms. Duringhomologous recombination, the ends of the targetingvector are removed (Capecchi, 1989). In contrast,random integration usually occurs at the ends of thevector, leading to insertion of the negative marker inthe genome. Cells that have undergone randominsertion can be rapidly eliminated by the additionof a prodrug. The most frequently used negativeselectable marker encodes HSV-TK, whose activityis similar to eukaryotic thymidine kinase. Unlikethe eukaryotic isoform, HSV-TK phosphorylatesgancyclovir and its relatives, which act as chain-terminating nucleoside analogs. Incorporation ofthese phosphorylated agents into DNA halts replica-tion and induces cell death. It is only the combinationof HSV-TK expression and presence of the prodrugthat is toxic to cells. Either component alone is rela-tively benign (Heyman et al., 1989). Addition of thisnegative selection step can increase the frequency ofidentifying appropriately recombined ES cells byapproximately 2000-fold (Mansour et al., 1988).

Rather than constructing a targeting vector de novo,it may be possible to obtain an appropriate vectorfrom the mutagenic insertion and chromosome engi-neering resource (MICER) repository, which hasindexed several thousand previously generated inser-tional targeting vectors. They can be used to disruptgenes, engineer insertions or deletions within a chro-mosome, or target both alleles of a gene within EScells (Adams et al., 2004). If an appropriate vector isavailable from the MICER resource, this can elimi-nate the challenging, labor-intensive development ofan efficient targeting vector.

Once built, targeting vectors are linearized andintroduced into ES cells by electroporation(Figure 6(b)). After a brief recovery period, thecells can be placed in positive selection media toidentify those that have integrated the targeting vec-tor into the genome into positions where the resis-tance gene can be expressed, that is, downstream of apromoter and into an exon. This is followed by thenegative selection media. This second step is meantto eliminate all cells into which the DNA has ran-domly integrated, leaving only homologous recombi-nants. While this is the ideal outcome, the targetingvector can become disrupted during the integration


process, losing the negative selectable marker. Hence,ES cell clones must be genetically evaluated to deter-mine which clones have, indeed, undergone homolo-gous recombination. This involves isolating DNA andperforming Southern blots with probes specific forthe integrated DNA as well as the targeted, endoge-nous sequence. After this validation step, the integrityof the genome must also be confirmed. Cells withgross chromosomal changes can arise followingclonal selection and these cells will not be able topopulate various lineages of the mouse. Karyotypingcan be used to eliminate aneuploid clones. When anES cell clone has passed these various criteria, it isthen propagated to produce sufficient cells for gen-erating chimeric mice.

85.3.1.2 Production of chimeric mice

The overall goal of genetically manipulating ES celllines is producing mice with a gene disruption.The transition from a clonal cell line to the acquisi-tion of genetically altered animals requires that theES cells be placed in an environment where they cancontribute to the formation of a mouse. This envi-ronment is a donor blastocyst, which is collected froma pregnant female. The ES cells are injected into theblastocele and will incorporate into the inner cellmass, which forms the embryo. Following injectionthe blastocysts are placed into a recipient female thatis hormonally primed to carry the embryos to term.During development, the two populations of cells inthe inner cell mass will populate portions of thevarious tissues of the mouse embryo, ultimately gen-erating a chimeric animal. Some cells in the mousewill be derived from the ES cell clone while otherswill be descendents of the originating blastocyst.Identifying chimeric mice is relatively straightfor-ward because the donor blastocyst is usually from adifferent genetic strain than the ES cells, with eachgenerating a different coat color. Thus, chimeric micehave patches of different color fur. While this isuseful for determining that the injection process waseffective and that ES cells populated the skin of themouse, the most important outcome is population ofthe germ line by the ES cells. If the ES cells con-tributed to the germ line, genetically altered progenycan be obtained from this chimera, making a new lineof mice. Alternatively, if the ES cells did not populatethe germ line, the chimeric animal has limited utility,being restricted to phenotypic analysis of a complexanimal without proper controls.

ES cell contribution to the germ line can be rap-idly assessed by breeding the chimeric mouse with a

mouse from the same genetic strain as the ES cell line(usually 129). Most ES cell lines are genetically male(XY genotype) and only male chimeras should beevaluated because a male ES cell line cannot produceviable oocytes. Progeny obtained from the chimerathat have the same coat color as the parental strainshould be derived from the ES cell line. These prog-eny are now genetically uniform, that is, all cells inthe animal have the same composition of genes. Incontrast, progeny that are a different coat color arederived from germ cells that originated from thedonor blastocyst.

If ES cells have contributed to the germ line of achimera, this mouse can be used to begin generating acohort of heterozygous null animals. At this point, itis important to carefully select the strain of mice towhich the chimera should be bred. Although most EScell lines correspond to the 129 strain, these animalshave low fecundity and are not particularly useful forbehavioral research due to their anatomical braindeficits (see Secti on 85 .1.2). Thus, the altered geneticallele must be moved to another genetic backgroundby creating congenic mice. This requires ten or moregenerations of backcrossing to the desired strain.During this transition, progeny can be used for vari-ous experiments; however, the interindividual varia-bility may be high due to differences in straincontributions in each animal. With each cross, theprogeny are genotyped for the presence of the dis-rupted allele using Southern blots or PCR. It is wiseto perform Southern blots with the first few genera-tions of mice to ensure that the targeted allele has notundergone an unexpected rearrangement and thatthe gene of interest is truly disrupted. The largenumber of generations that are required to producecongenic mice requires a significant investment oftime and resources. This process can be acceleratedby a speed congenic method that is guided by the useof genomic markers to select progeny that are genet-ically most similar to the destination strain (Markelet al., 1997). These progeny are then used forsubsequent crosses rather than randomly selectinganimals that may be more dissimilar. This approachreduces the number of backcross generations to fouror five (Wong, 2002). Recent improvements in manip-ulating C57BL/6 ES cells and embryos may subse-quently increase their future use for the productionof knockouts. This strain is more useful than 129 micedue to its high fecundity and normal brain structure;thus, these advances may assuage the typical delayassociated with production of congenics (Mishina andSakimura, 2007).


85.3.1.3 Generation of knockout animals andpedigree analysis

When heterozygous null animals are obtained in thedesired genetic strain, they must be intercrossed togenerate knockout animals. If the targeted allele isnot essential for life, it should follow a Mendelianinheritance pattern with a ratio of 1:2:1 (homozygouswild-type:heterozygous null:homozygous null). Witha sufficiently large cohort of mice, a skewed ratio isindicative of a developmental defect induced bythe mutant allele. The most obvious of these is aninability to obtain homozygous null (knockout) mice.Although the gene may not have a suspected rolein development, the absence of homozygous nullmice indicates that the gene plays a critical role atsome point between conception and early postnatallife. While not the anticipated result, this outcomeopens new avenues for characterizing the candidategene’s physiological function. Identifying the timeframe for death of knockout mice is the next stepfor determining what the unknown function mightbe. Usually, an analysis of mid- and late gestationpups can generate significant information. If the lategestation pups include knockout animals, this sug-gests that the gene disruption interferes with postna-tal life. In contrast, if no knockout pups can be foundat mid-gestation, an early embryogenesis or implan-tation defect is suspected.

Rather than complete lethality, it is also possiblethat homozygous null mice can be obtained, but inlower numbers than anticipated. Consistency ofskewed genotypes over a large number of litters indi-cates that the phenotype is only partially penetrantand loss of target-gene function is incompatible withlife in some animals and not others. This variabilitycan be due to several causes, but the most likely isthat the genetic background is still heterogeneousacross the population of animals being studied, thatis, the mice are not yet congenic. In this case, differ-ent alleles from the distinct genetic backgroundswould have varying contributions to the overall phe-notype. These are known as modifying genes becausethey modify the phenotype. Phenotypic variabilityshould be resolved by further backcrossing into thedesired strain. This may increase or decrease the rateof lethality depending on the contributions of thedestination strain. Once a consistent phenotype isobserved, it may be useful to identify the modifyingloci. This can be accomplished by crossing congenicswith various other strains and using genome scans toidentify markers that are in linkage disequilibriumwith the phenotype.

In a litter lacking homozygous null mice, thenumber of heterozygotes should be roughly twicethe wild-type progeny (a 1:2 ratio). On the otherhand, loss of just one allele may also affect an essen-tial developmental process. If so, this ratio will also beskewed. For most genes, a single copy is sufficient fornormal function and compensatory changes may evenresult in normal cellular levels of protein expressionfrom that one allele. However, some genes displayhaploinsufficiency, meaning that a single allele isincapable of producing a normal phenotype. Theextent to which gene dosage induces a particularphenotype is due to a complex array of factorsincluding the expression level of the protein fromthe remaining allele, the potential for haploinsuffi-ciency in the parent to affect the phenotype ofthe progeny, and the presence of genetic modifiers.Phenotypes of heterozygous mice can thus rangefrom being identical to wild-type mice to recapitu-lating the knockout. Where the heterozygotes fall inthis continuum is dictated by the specific gene beingknocked out. For example, disruption of both allelesof the Na,K-ATPase a3 gene induces early postnataldeath while heterozygous null animals are viable andfertile (Moseley et al., 2007). The heterozygotes, nev-ertheless, are not identical to wild-type mice. Loss ofone allele of this gene induces deficits in spatiallearning/memory, increased locomotor activity, andan increased response to methamphetamine. It hasbeen suggested that these mice, as well as those target-ing the other a-isoforms of this protein, may provideuseful animal models for evaluating the impact ofhaploinsufficiency of the Na,K-ATPase a3-isoform inhuman CNS disorders such as Alzheimer’s disease,where a decrease in isoform expression is alsoobserved (Chauhan et al., 1997; Hattori et al., 1998).

An important issue to consider when performingstudies with knockout mice is the selection of appro-priate controls. If the null mice are generated by inter-crossing heterozygotes, the litters will also includewild-type mice at roughly the same frequency as thehomozygous null animals. The wild-type littermatesrepresent the ideal control because they are matchedto the knockouts in age, genetic background, andenvironmental conditions. With this breeding scheme,a large number of heterozygotes are also produced. Ifthey have the same phenotype as wild-type animals,the two groups are usually combined to increase thepopulation size of the study. If a large number ofknockout mice are necessary, and inactivation of thegene has no impact on fertility, additional null micecan be generated by crossing two knockouts. This will


only generate knockout mice, with no wild-typecontrols and, this may be appropriate dependingon the goals of the experiment. For example, if thestudy focuses on the effects of a drug in the nullbackground, the experimental paradigm requiresonly null mice � treatment. Alternatively, if theimpact of the gene is the primary question, thenwild-type controls are essential. Mice from otherlitters can be used with the caveat that they may notrepresent accurate controls due to differences in age,genetics, and environment.

85.3.2 Knockin Mice

Phenotypes in knockout mice are usually the resultof completely eliminating the function of a gene.Embarking upon detailed mechanistic studies thatevaluate the functional significance of domains andmutations requires approaches where more modestchanges can be made to the protein. Transgenic micecan be used to determine the outcome of expressinga mutant protein; however, this method usuallyinvolves overexpression and can suffer from incorrecttemporal or spatial expression. A more effectiveapproach involves replacing endogenous genes withgenetic variants (i.e., knockin), which can take manyforms. Point mutations can be introduced into a geneto assess the impact of individual amino acids on genefunction and phenotype. Alternatively, a murine genecan be replaced by the human ortholog, producinghumanized mice. This can generate a clinically rele-vant in vivomodel for drug testing because the humanreceptor can be placed into the physiological contextof a whole animal. Substitutions of entire genes canalso be made between different members of a genefamily. This is particularly useful for assessing func-tional redundancy of highly related proteins. Lastly,knockin methodology can be used to create fusionproteins that are expressed with the same character-istics as the endogenous gene. For example, a GFPcassette can be inserted into either terminus of thetarget protein, generating a functional fusion proteinthat is readily visible in living animals using noninva-sive imaging or in individual cells with fluorescencemicroscopy, opening avenues for evaluating changesin subcellular localization, in vivo. All of theseapproaches evaluate more subtle changes in theprotein rather than its complete elimination. Theknockin method is ideal for determining the impactof changing a small portion of a gene, such as aspecific protein domain, without altering other fun-damental aspects of its expression or regulation.

Knockin mice require the same general methodsas knockouts, except that two rounds of recombina-tion are necessary in ES cells. As with the knockout, atargeting vector is constructed with positive and neg-ative selection markers. The positive marker is inter-nal to the targeting cassette and is flanked by loxPsites (see Secti on 85. 2.3.2). The negative marker is oneither the 3 0 or 5 0 end of the vector. The fir st rou nd ofrecombination swaps the endogenous sequence withthe targeting cassette, which in this case, also includesthe variant allele to be inserted. Once ES cells haveundergone the standard selection scheme, the pres-ence of the variant allele and the positive selectablemarker in the targeted locus is confirmed withSouthern blots. These ES cell clones are now sub-jected to a second round of recombination to removethe positive selectable marker via recombinase-mediated cassette exchange (RCME; Gu et al., 1993;Zou et al., 1994). By transiently transfecting theclones with a Cre-recombinase expression vector,the region between the loxP sequences (i.e., the posi-tive selectable marker) will be excised and lost. Theend result is a modestly modified allele that containsa loxP sequence and no selection cassettes. To furtherminimize the effects of the knockin process, the loxPsites can be engineered into an intron in the targetgene. Assuming that the intron is largely nonfunc-tional, this should have minimal effects on theexpression of the gene.

Knockin technology began with studies that eval-uated the regulatory regions of the IgH locus (Guet al., 1993) and were rapidly followed by productionof mice that express humanized antibodies (Zou et al.,1994). Since then, many groups have used knockins toaddress questions about the functions of variousregions of targeted proteins within a physiologicalcontext. A particularly informative example is theuse of this technology to disrupt the DNA-bindingdomain of the glucocorticoid receptor (GR). Thisreceptor regulates transcription through two primarymechanisms: it can bind to DNA and regulate tran-scription directly or it can bind to other transcriptionfactors and modulate their function. Knockin meth-odology was used to discern the relative importanceof these two activities by disrupting the DNA-binding domain of the receptor while maintainingthe rest of the endogenous protein’s structure. Althoughcomplete disruption of the GR gene induces earlypostnatal death due to severe congenital atelectasisand respiratory failure (Cole et al., 1995), mice with asingle-point mutation that disrupts DNA binding(GRdim) are viable and fertile. This study led to the


surprising conclusion that many actions of GR do notrequire DNA binding (Reichardt et al., 1998). Of note,these mice displayed deficits in spatial memory, buthad normal locomotion, exploratory, and anxiety-associated behaviors (Oitzl et al., 2001). Hence,DNA binding by GR is necessary for regulatingsome behaviors and not others. This was further rein-forced by a model in which the GR was knocked outonly in the ner vous system (GR NesCre ) ( see Secti on85.3.3 for meth od description ). In contrast to theDNA-binding mutation, complete absence of GRin the nervous system reduces anxiety (Troncheet al., 1999).

85.3.3 Conditional Gene Targeting

According to the National Human Genome ResearchInstitute, approximately 15% of all gene knockoutsdisplay developmentally lethal phenotypes, meaningthat these animals cannot grow to adulthood andproduce progeny. In these situations, phenotypic ana-lyses are confined to the embryo or neonate, whichmay be perfectly acceptable if analyzing developmentis the ultimate goal. However, if the goal is to assess theimpact of gene disruption on behavior, developmentallethality poses an insurmountable hurdle. Althoughembryonic lethality is an extreme outcome, it mustbe emphasized that all knockout mouse phenotypesare a conglomeration of all of the cellular changesthat occur in response to the loss of a gene. The

21

ne2

Targeting vector

Targeted gene

1

21

Colpro

Floxed allele

Cros

Tissue with Cre expression

31

Nonfunctional allele

neor

Figure 7 Conditional gene targeting. The targeting vector contselection marker (TK) on the end, and two loxP motifs (black tria

will excise the region between the loxP sequences, while cells t

fundamental remodeling of homeostatic processesthat ensues impedes assigning cell-autonomousmechanisms to that gene. In addition, developmentalchanges may be evoked that allow highly related genefamily members to compensate for the loss of agene. All of these contingencies can be addressed bythe production of conditional knockoutmice. An illus-trative example again involves GR. Genetic variants ofGR have been associated with major depressive disor-der (van West et al., 2006; van Rossum et al., 2006),suggesting that it may play an important role in reg-ulating disease susceptibility. Developing animalmodels to study the role of this receptor in modulatingdepressive behaviors should generate insights intomechanisms and therapeutic interventions. As dis-cussed above, germ-line disruption of GR causesearly postnatal death (Cole et al., 1995). Hence, exam-ining the impact of GR on behavior requires alterna-tive methods. Some studies have used overexpressionof the receptor in specific regions of the brain(Wei et al., 2004) or targeted antisense RNA expres-sion (Pepin et al., 1992) to suppress GR levels, butthese approaches can suffer from excessive overex-pression or incomplete knockdown, respectively.These studies are complemented by tissue-specificgene disruption (i.e., conditional knockout) wherethe GR gene is inactivated in specific regions of thebrain but remains functional in the rest of the bodyand has supported a brain-specific role for GR inregulating anxiety (Tronche et al., 1999).

3

or

3 TK

3

lection of ES cell clonesduction of mice

s with Cre transgenic

Tissue without Cre

321

Functional allele

neor

neor

ains a positive selection maker (neor ) in an intron, a negativengles) flanking the second exon. Any cells that express Cre

hat do not express Cre will maintain a functional allele.


Conditional gene targeting utilizes Cre-mediatedgene recombination in a manner similar torecombination-mediated inducible transgene expres-sion (Secti on 85.2.3 .2). For conditio nal knockouts, theloxP sequences flank an essential region of an endog-enous gene rather than a randomly inserted trans-gene. These sites are placed into the gene of interestusing a knockin approach. If the loxP sequences arein the same orientation, Cre will excise the portion ofthe gene that resides between them. To avert thepossibility that the loxP sequences alone may disruptthe gene, they are usually placed within introns thatsurround an essential coding exon (Lewandowski,2001). With this configuration, the gene should func-tion normally until a Cre-mediated recombinationevent has transpired. To induce recombination,mice with the floxed allele are bred with another setof transgenic animals that express Cre tissuespecifically. A breeding paradigm is then used togenerate compound transgenic mice that are homo-zygous for the floxed allele with the Cre transgene.Only cells that express Cre will undergo the recom-bination process and delete the region of the targetedgene that resides between the loxP sequences. As analternative to transgenic delivery of Cre, floxed micecan be injected with adenoviruses that express therecombinase (Rohlmann et al., 1996). While rapid,this approach can be limited by the accessibility ofthe cells of interest for injection and the potential forpoor infection efficiency.

Insertion of loxP sequences into a gene requiresbuilding an appropriate targeting vector that isvery similar to the type used for conventional knock-outs, except that Cre-recognition motifs (loxP) areincorporated to provide a means for removing thepositive selection cassette (Figure 7). Positive andnegative selection cassettes are included to permitisolation of ES cell clones with a high likelihood ofundergoing homologous recombination between thetargeting vector and the endogenous gene. Whileconventional targeting usually uses a positive selec-tion marker to disrupt the coding sequence of thegene, conditional targeting requires that the markerhas no impact on normal gene activity because thegene must function properly in all cells that have notundergone Cre-mediated recombination. To fulfillthis criterion, the marker is usually placed in anintron of the targeted gene. Unless this intron isnecessary for proper regulation of the gene, thisshould permit normal activity. To completely avertthe possibility that the selection marker may disruptgene function, it can be removed after isolating clonesthat have the desired loxP architecture. This can be

accomplished by flanking the selection cassette withrecognition sequences for another recombinase suchas FLP. Transient transfection of ES cells with a FLPexpression vector will induce the excision of theselectablemarker (Schaft et al., 2001). The appropriateconfiguration of targeted allele within clones shouldthen be validated by PCR or Southern blotting.

Following confirmation of the genetic composi-tion of the floxed ES clones, these cells are usedto produce chimeric mice (and subsequently hetero-zygous animals) by applying the same protocols as astandard knockout. These mice should be phenotyp-ically identical to wild-type animals. At this point,two subsequent breeding rounds are necessary toobtain animals with cell-specific, homozygous genedisruption. Heterozygous floxed mice are firstcrossed with Cre-expressing transgenics to generatecompound heterozygotes containing one floxed alleleof the targeted gene as well as the Cre-expressingtransgene. In these animals, the single floxed allelewill be excised by Cre while the other allele willremain intact. This should not present a major hurdleunless the gene is haploinsufficient. In that case,an inducible Cre transgene will be necessary (seeSecti on 85.2.3) . Assumi ng tha t the compou nd hetero-zygotes (Creþ flox) are viable and fertile, they arebackcrossed to heterozygous (flox) mice to garnerprogeny that have two floxed alleles plus the Cretransgene. The Cre recombinase will excise theregions between the loxP sequences in both alleles,resulting in a knockout for that gene in any cell that iscapable of expressing Cre.

In most cases, obtaining a measurable phenotyperequires disruption of a gene in a large percentage oftargeted cells (Sauer, 1998; Lewandowski, 2001). Thiscan sometimes be difficult to achieve due to variableexpression of the Cre recombinase and the hetero-chromatin environment of the targeted transgene.The likelihood of complete ablation of the targetgene can be increased by using a compound knockoutmouse where one allele has undergone conventionalknockout while the other allele is floxed. Thismethod increases the probability that a productiverecombination event will occur because Cre-mediated disruption is only required for one allelein each cell rather than two.

Application of Cre-mediated recombination can behampered by the limited availability of promoters thatconvey the desired temporal pattern of Cre expres-sion. If a significant delay exists between the onset ofCre expression and phenotyping, it is possible that theanimal will have undergone compensatory processesthat alter the phenotype. Compensation can occur


through several routes. The most straightforwardmechanism is upregulation of protein-family mem-bers that have similar functions as the disruptedgene. This is a substantial concern when studying anorgan with the degree of plasticity as the brain withsome knockout phenotypes being misleading due toadaptations during development. For this reason, it ishighly desirable to disrupt genes at predeterminedtimes, for instance, after a specific developmental win-dow. The same inducible approaches for regulatingtransgene expression described above (see Sections85.2.3.1 and 85.2.3.2) can be used to control the timingof Cre expression or activity. Alternatively, Creexpression/activation can be precisely timed by exog-enous delivery of the recombinase by viral infection.Injection of a Cre-expressing virus into a specifictissue will largely confine Cre to that region, althoughthe ability to target all cells within the tissue is gener-ally lacking. This approach has recently been used inmice containing two floxed alleles of the brain-derivedneurotrophic factor (BDNF) gene. Injection of a Cre-expressing adeno-associated virus into the nucleusaccumbens resulted in highly localized loss of BDNFexpression. This approach ultimately revealed thatBDNF in this brain region plays an important rolein regulating cocaine self-administration (Grahamet al., 2007).

85.4 Gene Trapping

In addition to targeted gene disruption, ES cells canalso be used to randomly mutagenize the genome,either by chemical or genetic means. The goal isto disrupt as many genes as possible, anticipatingthat at least some will produce a desired phenotype.Gene trapping is a common genetic method of muta-genesis that provides a mechanism for cloning (i.e.,trapping) disrupted genes from animals with a phe-notype. It provides a major advance over chemicalmutagenesis where disrupted genes must be identi-fied by conventional marker-assisted linkage analysis.A high-throughput, single vector is used for genetrapping. These vectors can take a variety of forms,but the basic principle is the same with endogenousgenes being disrupted by a cassette that encodes adominant selectable marker (e.g., neor, or the lacZ/neor fusion: bgeo; Stanford et al., 2001). Modificationscan be made to the vector to tailor it to a specificpurpose or screen.

Promoter trap vectors contain the coding sequencefor a selectable marker upstream of a polyadenylationsequence. When this sequence is integrated into an

exon, the polyadenylation site will truncate thatgene’s mRNA, preventing production of a full-lengthprotein. Often, this produces a completely nonfunc-tional allele. Alternatively, it may result in a truncatedprotein that retains some, if not all, of its function,potentially producing a hypomorphic allele. Theselection cassette in these vectors lacks a promoter;thus, it can only be expressed and convey antibioticresistance if it is inserted downstream of a transcrip-tional start site. Hence, by placing cells in selectionmedia, only those that have undergone an integrationevent within a transcribed region will survive. Inlater-generation vectors, an internal ribosome entrysite (IRES) was engineered upstream of the selectioncassette to permit translation that is independent ofthe targeted gene’s reading frame. Individual integra-tion sites (i.e., disrupted genes) in clones can be rap-idly identified using the selectable marker as apriming site for amplifying and sequencing the gene.It is important to note that the design of this systemnecessitates that the targeted gene be expressed in theES cells, because the selection cassette does not con-tain its own promoter. If the genes of interest are notexpressed in these cells, alternative methods must beused for mutagenesis (Lee et al., 2007).

As stated above, the goal of gene trapping is torapidly mutagenize most, if not all, genes in thegenome. By selecting clones and identifying theirintegration sites, a library can be produced that con-tains individually trapped ES cell lines, each withdistinct gene disruptions. Several large gene trappingprograms have joined to form the InternationalGene Trap Consortium (IGTC). This conglomeratemakes the vast number of characterized, targeted EScell lines publicly available through a web portal(Nord et al., 2006). In late 2005, the library of clonedcell lines included�40% of known mouse genes withthe objective of obtaining clones that cover most, ifnot all, genes. In the near future, knockout cell linesfor nearly any gene of interest should be readily avail-able from the consortium. This eliminates the com-plexities associatedwith designing individual targetingvectors as well as screening ES cell clones. Rather,the entry point into producing mice with a targetedgene disruption becomes ordering a selected ES cellline with a trap in that gene which can be injected intoblastocysts at the investigator’s home institution.

85.5 Genetic Environments

A last caveat must be considered when deriving con-clusions regarding genotype:phenotype relationships


in genetically manipulated animals. All phenotypesare the products of vast networks of genetic interac-tions. Hence, any alteration of the genome in a mul-ticellular organism will generate primary andsecondary consequences, all of which contribute toa phenotype. In addition, the baseline on which thegenetic change was made will have an impact on theextent and composition of the phenotype. As a resultof this extensive interconnectivity, all phenotypes areultimately the sum of the entire genetic environment,not just the changed gene.

85.5.1 Genetic Background andPhenotypes

Phenotypes of mouse models can vary dramaticallydepending on the strain and can range from embry-onic lethality in one strain to full viability and fertil-ity in another (LeCouter et al., 1998; Nadeau, 2005;Ni et al., 2007). Genetic background or strain is also amajor modulator of murine behaviors, includingthose observed in transgenic or knockout mice. Thepowerful impact of genetic background on pheno-typic outcomes was first revealed when disruptionof the epidermal growth factor receptor (EGFR)resulted in a wide range of pathologies in differentstrains of mice (Threadgill et al., 1995; Sibilia andWagner, 1995). Strain-dependent phenotypic incon-gruity arises due to the variable input of modifiergenes that regulate the manifestation of individualphenotypes. Modifier genes are epistatic to the tar-geted gene, that is, they have a genetic interactionthat affects the phenotype. This can be directly atthe level of the epistatic gene (e.g., the product ofone gene may regulate the expression of another), orit may be indirect (e.g., their products may bothparticipate in a complex pathway). Modifiers mayalso impact one phenotype (e.g., cancer susceptibi-lity) and not another (e.g., fertility) in the samegenetically manipulated model because the pheno-types are the consequences of perturbations in dis-tinct pathways.

While the influence of genetic background can beviewed as a confounding variable when using geneti-cally engineered animals, strain-dependent shifts in aphenotype can be used to unveil new pathways thatintersect with the disrupted/manipulated gene. Themodifying genes can be identified using marker-assisted linkage analyses. While labor intensive, andnot the initial goal of the research plan, discoveringand characterizing these modifiers can lend tremen-dous insight into the mechanisms that contribute to

the phenotype (MacPhee et al., 1995; Hide et al.,2002). In particular, novel pathways that interactwith the gene/protein of interest and modulate dis-ease pathologies can be revealed and this informationused for rational drug design.

85.5.2 Hitchhiking or Passenger Genes

Most mouse ES cell lines are derived from the 129strain. Given its limitations, most knockouts begin inthe 129 background, but congenic mice are rapidlyproduced where the disrupted allele is movedinto the genetic background of another strain (seeSecti ons 85.1.1 and 85.3.1.2). By vir tue of sister chro-matid exchange during each round of gametogenesis,even the chromosome that harbors the altered allelewill eventually transition to being predominantlyderived from the destination strain. However, recom-bination will rarely occur immediately proximal tothe altered gene. Consequently, the genes nearestto the targeted allele will still be from the parentalstrain, that is, the ES cell strain, rather than thedestination strain. Even with 12 generations of back-crossing, approximately 1% of the genome surround-ing the targeted allele will correspond to the parentalstrain. Depending on the genetic density of the chro-mosomal region, this could include hundreds ofgenes (Lathe, 1996; Gerlai, 1996), which are calledhitchhiking genes because they always accompanythe targeted allele to any destination genetic back-ground (Gerlai, 1996). While most of these geneswill be irrelevant to the phenotype, some may beimportant modifiers. In extreme situations, theappearance of the phenotype can be due solely toaltering the contextual background of a hitchhikinggene rather than the targeted genetic modification(Kelly et al., 1998).

When using transgenic approaches, the possibilitythat hitchhiking genes are the underlying cause of aphenotype can easily be negated if mice representingmultiple transgene integration sites are examined.Each integration site will affect distinct genomicregions; hence, a consistent phenotype spanning atleast two lines indicates that the transgene was thekey determinant of the phenotype. It is more chal-lenging to address this issue in knockouts becausethe integration site is identical for all mice. Numer-ous approaches have been proposed. The most obvi-ous involves directly testing Koch’s postulate: ifremoving the function of a gene induced a pheno-type, then restoring activity should cause its reversal.Transgenic mice are produced that express the


wild-type form of the targeted gene under control ofa ubiquitous promoter in the same genetic back-ground as the knockout. If the transgene reversesthe phenotype, the targeted gene was the underlyingcause. A more complex method to address this ques-tion is to produce mock knockout mice, where anirrelevant marker is inserted into the same position ofthe chromosome but does not disrupt the target gene.If congenics derived from this marker strain have thesame phenotype as the knockout, this implicatesthe surrounding hitchhiking genes rather than thetargeted allele. Both approaches are relatively resourceintensive. A more cost-effective approach that hasmultiple experimental uses involves producing a con-ditional knockout animal that has loxP sequencesinserted into the gene. Animals with and withoutrecombination can then be examined. If animalswithout a Cre transgene have a phenotype, the sur-rounding genes are again indicted.

85.6 Summary

Development of approaches to manipulate themurine genome has greatly expanded our capabilityto complete detailed mechanistic studies of endocri-nology and behavior. This has led to a transitionwhere mice are becoming a vital model for studyingthe genetic basis of various behavioral characteristics,although further development of appropriate behav-ioral tests for this species is imperative. While eachmethod for altering the genome faces some caveats,the use of genetically manipulated mice has revolu-tionized our understanding of the molecular/geneticevents that control higher brain function. Further,these approaches have spawned novel models ofhuman disease and instigated the production of ani-mals where molecular processes could be directlyvisualized in the brain. More recent advances in ratgenomics and genetic engineering hold promise forbringing this established model of human behaviorinto the age of genetic dissection as well. It is antici-pated that use of genetic methodology will continueto grow and revolutionize our understanding of theprecise interplay between genes and their environ-ment in regulating diverse arrays of behavior.

Acknowledgment

The authors thank Darcie Seachrist for thoughtfuldiscussions regarding the content of this chapter.

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Relevant Websites

http://www.informatics.jax.org – Mouse Genome Informatics.http://nobelprize.org – Official website of the Nobel Foundation.

http://www.informatics.jax.org

http://nobelprize.org

Biographical Sketch

Ruth A. Keri, PhD, received her doctorate in pharmacology from Case Western Reserve University School of Medicine andcontinued her postdoctoral studies in the same department. Her work during this time focused on the use of transgenic

mouse models to define the regulatory mechanisms underlying expression of the gonadotropin subunit genes. She iscurrently an associate professor in the Department of Pharmacology at Case Western Reserve University in 1999 and herlaboratory studies mechanisms of breast development and carcinogenesis using a combination of in vitro models as well as

transgenic and knockout mice. She was the director of the CWRU Institutional Transgenic Mouse Core Facility from 2000to 2003 and has sat on institutional and national advisory committees regarding the use of animal models for variousdiseases.

Ruth E. Siegel, PhD, received her doctorate in pharmacology fromHarvardMedical School. She then performed postdoctoralstudies concerning neurotransmitter expression and regulation at Harvard Medical School and the National Institute ofMental Health. She is currently a professor in the Department of Pharmacology at Case Western Reserve University where

she has been working since 1986. Her research interests are focused on the developmental expression and regulation ofGABA-A receptor subunit genes. Her studies have demonstrated that receptor subunit expression is plastic and that subunitsin different brain regions are differentially regulated in response to environmental cues.

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Hormones, Brain and Behavior || Transgenic and Genetic Animal Models