BioSci 145B lecture 9 page 1 © copyright Bruce Blumberg 2004. All rights reserved BioSci 145B Lecture #9 5/31/2005 Bruce Blumberg –2113E McGaugh Hall -

BioSci 145B lecture 9 page 1 ©copyright Bruce Blumberg 2004. All rights reserved

BioSci 145B Lecture #9 5/31/2005

• Bruce Blumberg– 2113E McGaugh Hall - office hours Wed 12-1 PM (or by appointment)– phone 824-8573– [email protected]

• TA – Suman Verma [email protected]– 2113 McGaugh Hall, 924-6873, 3116

DON’T FORGET – TERM PAPERS ARE DUE BY 5 PM on Thursday

June 2

• Suman will not be here this week so please either – drop the paper off to me or – submit online using the drop box or– E-mail it to me. The time stamp on your mail

will determine whether it is on time or not

mailto:[email protected]

mailto:[email protected]


Conditional gene targeting

• Many gene knockouts are embryonic lethal– some of these are appropriate and expected

• gene activity is required early– others result from failure to form and/or maintain the placenta

• ~30% of all knockouts• Clearly a big obstacle for gene analysis

• How can this be overcome?– Generate conditional knockouts either in particular tissues or

after critical developmental windows pass– Sauer (1998) Methods 14, 381-392.


Conditional gene targeting - contd

• Approach– recombinases perform

site-specific excision between recognition sites

– FLP system from yeast• doesn’t work well

– Cre/lox system from bacteriophage P1

• P1 is a temperate phage that hops into and out of the bacterial genome

• recombination requires – 34 bp recognition sites

locus of crossover x in P1(loxP)

– Recognized by Cre recombinase

• if loxP sites are directly repeated then deletions• if inverted repeats then inversions result


Conditional gene targeting (contd)

• Strategy– Make targeting construct

(minimum needed for grant)– homologous recombination,– transfect CRE, select

for loss of tk– Southern to select

correct event• Result called

“floxed allele”– inject into blastocysts,

select chimeras– establish lines – cross with Cre expressing

line and analyze function



– Tissue- or stage-specific knockouts from crossingfloxed mouse with specific Cre-expressing line

– requirement for Cre lines

• must be well characterized

– promoters can’t be leaky

• Andras Nagy’s database of Cre lines and other knockout resources http://www.mshri.on.ca/nagy/cre.htm

http://www.mshri.on.ca/nagy/cre.htm

http://www.mshri.on.ca/nagy/cre.htm



• advantages– can target recombination to specific tissues and times– can study genes that are embryonic lethal when disrupted– can use for marker eviction– can study the role of a single gene in many different tissues with

a single mouse line– can use for engineering translocations and inversions on

chromosomes

• disadvantages– not trivial to set up, more difficult than std ko but more

information possible– requirement for Cre lines

• must be well characterized regarding site and time of expression

• promoters can’t be leaky (expressed when not intended)


Genome wide analysis of gene function

• How to mutate all genes in a given genome?– Easy with microbial genomes – can mutate all yeast genes by

homologous recombination– Recombine in selectable marker– Propagate strain and analyze phenotypes

Target gene

Selectable marker(antibiotic resistance)

Homology region

Unique oligonucleotide“barcodes” for PCR


Genome wide analysis of gene function (contd)

• How about gene targeting in other organisms– With more complex genomes and more genes?– Huge undertaking to specifically target 30K+ genes in

mammalian cells• Difficulty• Expense• Inability to target all possible loci

– Some efforts to make mouse collection • Lexicon Genetics has a collection of ES cells

– Drosophila collection as well– Driving force behind these efforts is

• Genome annotation• Drug target discovery (Lexicon)• Functional analysis


Genome wide analysis of gene function (contd)

• Main method for gene targeting in more complex organisms is random insertional mutagenesis– Transposon mutagenesis

• Bacteria – Tn transposons• Yeast - Ty transposons• Drosophila - P- elements• Vertebrates - Sleeping Beauty transposons

– Viral infection• Typically retroviruses – host range selectivity is obstacle

– Gene or enhancer trapping – modified viruses or transposons


Insertional mutagenesis - Gene trapping

• enhancer trap is designed to bring inserted reporter gene under the control of local regulatory sequences– put a reporter gene adjacent to a weak promoter (enhancer-less),

e.g. a retrovirus with enhancers removed from the LTRs– may or may not disrupt expression – Hopkins zebrafish group used unmodified virus

• viruses and transposable elements can deliver DNA to random locations– can disrupt gene function – put inserted gene under the

control of adjacent regulatory sequences

– BOTH


Insertional mutagenesis - Gene trapping (contd)

• enhancer trap (contd)– expression only when integrate into an active transcription unit

• reporter expression duplicates the temporal and spatial pattern of the endogenous gene

– reporters used -gal was the most widely used reporter• GFP is now popular -lactamase is seeing increasing use

– advantages• relatively simple to perform• active promoters frequently targeted, perhaps due to open

chromatin– Disadvantages

• Inactive promoters probably not targeted • insertional mutagenesis not the goal, and not frequent

– overall frequency is not that high• relies on transposon or retroviruses to get insertion

– may not be available for all systems, requires transgenesis or good viral vectors


Insertional mutagenesis – Gene trapping (contd)

• expressed gene trap (many variations possible)– goal -> ablate expression of endogenous gene, replace with transgene– Make insertion construct – reporter, selection, polyA sites

• No promoter but has a splice-acceptor sequence 5’ of reporter• Can only be expressed if spliced into an endogenous mRNA

– Transfer into embryonic cells, generate a library of insertional mutagens

• Mouse, Drospophila, zebrafish, frog– reporter expression duplicates the temporal and spatial pattern of the

endogenous gene


Insertional mutagenesis - Gene trapping (contd)

• Expressed gene trapping (contd) – advantages

• insertional mutagen– gives information about expression patterns– can be made homozygous to generate phenotypes

• higher efficiency than original trapping methods• selectable markers allow identification of mutants

– many fewer to screen– dual selection strategies possible

– disadvantages• overall frequency is still not that high• frequency of integration into transcription unit is not high

either• relies on transposon or retroviruses to get insertion

– may not be available in your favorite system.– Uses

• Insertional mutagenesis• Marking genes to identify interesting ones• Gene cloning


Generating phenocopies of mutant alleles

• How to inactivate endogenous genes in a targeted but general way?– Important new development is RNAi – RNA

interference– Observation is that introduction of double-

stranded RNAs into cells lead to destruction of corresponding mRNA (if there is one)

– Principle is siRNA – small interfering RNAs– These generate small single stranded RNAs

that target mRNAs for destruction by– RISC – RNA interference silencing complex– First applied in C. elegans where it works

extremely well• Can introduce siRNA into cells even by

feeding to the worms!• Works very well in Drosophila• variably in mammalian cells• Poorly in Xenopus


RNAi (contd)

• Dicer complex generates short duplexes from dsRNA in the cell– Important to have 2-nt

overhangs• siRNAs are generated from these

fragments– Antisense strand binds to

mRNA and this recruits the RISC - RNAi silencing complex

– Complex leads to mRNA cleavage and destruction

• Two important reviews to read– McManus and Sharp (2002)

Nature Reviews Genetics 3, 737-747

– Dykxhoorn et al. (2003) Nature Reviews, Molecular Cellular Biology 4, 457-467


RNAi (contd)

• Micro RNAs are small cellular RNAs that previously lacked any known function– Always form a hairpin structure

with mismatches in stem• Turn out that micro RNAS direct gene

silencing via translational repression– (miRNAs) are mismatched

duplexes that dicer processes into stRNAs (small temporal RNAs)

– Use same cellular complex as siRNAs

– Perfect matches -> target cleavage

– Imperfect matches -> translational repression of target

• Two important new papers– Giraldez et al (2005) Science 308,

833-838 (microRNAs regulate brain morphogenesis)

– Lecellier et al (2005) Science 308, 557-560 (microRNA mediates antiviral defenses in human cells)


RNAi (contd)

• Parallels between siRNA and miRNA-directed RNAi


RNAi (contd)

• Ways to generate short RNAs that silence gene expression in vitro– a) chemical synthesis of siRNA, introduce into cell– b) synthesize long dsRNA, use dicer to chop into siRNA– c) introduce perfect duplex hairpin, dicer generates siRNA– d) make miRNA based hairpin, dicer generates silencing RNA

• Introduce into cells or organism by microinjection, transfection, etc.– Expression is transient– can only generate phenotypes for a short time after introduction


RNAi (contd)

• Ways to generate short silencing RNAs in vivo– Continuing expression to generate stable phenotype– a) produce long hairpin

from pol II promoter, letdicer make siRNA

– b) produce two transcriptsfrom pol III promoter, letanneal in cells

– c) produce a short hairpinfrom pol III promoter, letdicer generate siRNAs

– d) produce imperfecthairpin from pol II promoter, let dicergenerate miRNAs that direct gene silencing


RNAi (contd)

• RNAi for whole genome functional analysis– First generate library of constructs that generates siRNA or stRNA– Introduce these into cells, embyos (fly, frog, mouse) or animals

(C. elegans, plants)• For C. elegans, make the library in E. coli and simply feed

bacteria to worms• Must microinject or transfect with other animals

– Evaluate phenotypes


Antisense methods to knock out gene function

• Antisense oligonucleotides can transiently target endogenous RNAs– For destruction

• Many methods and oligo chemistries available• Most are very sensitive to level of antisense oligo, these are

degraded and rapidly muck up cellular nucleotide pools leading to toxicity

– For translational inhibition• Morpholino oligos appear to work the best

– Morpholine sugar is substituted for deoxyribose– Is not a substrate for cellular DNAses or RNAse H– Base-pairs with RNA or DNA more avidly than standard

DNA– The oligo binds to the area near the ATG in the transcript

and inhibits translation of the protein

– Deoxyribose morpholine

N

OO


Oligodeoxyribonucleotide Morpholino Oligonucleotide

B = A, C, T, GO

O

OB

O

O

P O

O

O

B

P O

O

O

N

O B

P N

O

O

N

O B

O

P N

O

O

Antisense methods to knock out gene function (contd)


Most Molecules Function in Complexes

• Given a target, how can we identify interacting proteins?

• Complex members may be important new targets– pharmacology– toxicology– Endocrine disrupter

action

• High throughput, genome wide screen is preferred– 20 years is too long


How can we approach whole genome analysis of protein complex formation?

• Each protein interacts with average of 3 others

• Many are much more complex

• Two papers this Thursday and one next Thursday describe two different approaches to this problem.


How to identify protein-protein interactions on a genome wide scale?

• You have one protein and want to identify proteins that interact with it

• You want to identify all proteins that interact with all other proteins

– straight biochemistry• Co-immunoprecipitation• GST-pulldown

– Library based methods• phage display• Yeast two hybrid• in vitro expression cloning

– Proteomic analysis– Protein microarrays– Large scale two-hybrid


Mapping protein-protein interactions• biochemical approach –

– what are some ways to purify cellular proteins that interact with your protein

– pure protein(s) are microsequenced

– advantage• functional approach• stringency can be manipulated• can identify multimeric proteins or complexes• will work if you can purify proteins

– disadvantages• much skill required• low throughput• considerable optimization required

• co-immunoprecipitation• GST-pulldown• affinity chromatography• biochemical fractionation


Mapping protein-protein interactions (contd)

• GST (glutathione-S-transferase) pulldown assay– Versatile and general– Fuse protein of interest

to GST– Incubate with cell or

tissue extracts– Mix with glutathione-

sepharose beads• Binds GST-fusion

protein and anything bound to it

– Run SDS-PAGE– Identify bands

• Co-Ip (immunoprecipitation) is identical except that antibody is used to pull down protein X



• scintillation proximity assay– Target is bound to solid phase –

bead or plate– radioactive protein or ligand is added

and allowed to reach equilibrium• 35S, 125I, 3H work best

– radioactive decay is quenched in solution, only detected when in “proximity” of the solid phase, e.g. when bound to target

– applications• ligand-receptor binding with 3H small molecules• protein:protein interaction• protein:DNA



• FRET - fluorescent resonance energy transfer– based on the transfer of energy

from one fluor to another that is not normally excited at that wavelength

– Many types of fluorescent moieties possible

• rare earth metals – europium cryptate

• fluorescent proteins – GFP and variants– allophycocyanin

• Tryptophan residues in proteins– application

• very commonly used for protein:protein interaction screening in industry

• FRET microscopy can be used to prove interactions between proteins within single cells

– Roger Tsien at UCSD is expert



• FRET (contd)– advantages

• can be very sensitive• may be inexpensive or not depending on materials• non-radioactive• equilibrium assay• single cell protein:protein interactions possible• time resolved assays possible

– disadvantage• poor dynamic range - 2-3 fold difference full scale• must prepare labeled proteins or ligands

– Not suitable for whole genome analysis• tunable (or multiwavelength capable) fluorometer required

(we have one here)



• Biacore (surface plasmon resonance)– surface plasmon waves are excited at a metal/liquid interface– Target bound to a thin metal foil and test sample flowed across it– Foil is blasted by a laser from behind

• SPR alters reflected light intensity at a specific angle and wavelength

• Binding to target alters refractive index which is detected as change in SPR

• Change is proportional to change in mass and independent of composition of binding agent



• Biacore (contd)– Advantages

• Can use any target• Biological extracts possible• Measure kinetics• Small changes detectable with correct instrument

– 360 d ligand binding to 150 kd antibody• Can use as purification and identification system

– Disadvantages• Machine is expensive (we have two)• “high throughput” very expensive• Not trivial to optimize


Library-based methods to map protein-protein interactions (contd)

• Phage display screening (a.k.a. panning)– requires a library that expresses

inserts as fusion proteins with a phage capsid protein

• most are M13 based• some lambda phages used

– prepare target protein• as affinity matrix• or as radiolabeled probe

– test for interaction with library members• if using affinity matrix you purify phages from a mixture• if labeling protein one plates fusion protein library and

probes with the protein– called receptor panning based on similarity with panning

for gold


Library-based methods to map protein-protein interactions (contd)• Phage display screening (a.k.a. panning) (contd)

– advantages• stringency can be manipulated• if the affinity matrix approach works the cloning could go

rapidly– disadvantages

• Fusion proteins bias the screen against full-length cDNAs• Multiple attempts required to optimize binding• Limited targets possible• may not work for heterodimers• unlikely to work for complexes• panning can take many months for each screen

– Greg Weiss in Chemistry is local expert



• Two hybrid screening– originally used in yeast, now

other systems possible– prepare bait - target protein

fused to DBD (GAL4) usual• stable cell line is commonly

used– prepare fusion protein library

with an activation domain - prey– What is the key factor required for

success?

– approach• transfect library into cells and either

select for survival or activation of reporter gene

• purify and characterize positive clones

• no activation domain in bait!



• Two-hybrid screening (contd)– Can be easily converted to

genome wide searching by making haploid strains, each containing one candidate interactor

– Mate these and check for growth or expression of reporter gene

Bait plasmid Prey plasmid

If interact, reporter expressed and/or

Yeast survive



• In vitro interaction screening - based on in vitro expression cloning (IVEC)– transcribe and translate cDNAs in vitro into small pools of proteins

(~100)– test for their ability to interact with your protein of interest

• EMSA• co-ip• FRET• SPA

– advantages• functional approach• smaller pools increase sensitivity• diversity of targets

– proteins, complexes, nucleic acids, protein/nucleic acid complexes, small molecule drugs

– very fast– disadvantages

• can’t detect heterodimers unless 1 partner known• expensive consumables (but cheap salaries)

– Typical screen may cost $10-15K• expense of automation

Documents

BioSci 145B lecture 9 page 1 © copyright Bruce Blumberg 2004. All rights reserved BioSci 145B Lecture #9 5/31/2005 Bruce Blumberg –2113E McGaugh Hall -