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Copyright � 2007 by the Genetics Society of AmericaDOI: 10.1534/genetics.106.065961
The Carnegie Protein Trap Library: A Versatile Tool for DrosophilaDevelopmental Studies
Michael Buszczak,* Shelley Paterno,* Daniel Lighthouse,* Julia Bachman,* Jamie Planck,*Stephenie Owen,* Andrew D. Skora,* Todd G. Nystul,* Benjamin Ohlstein,* Anna Allen,*
James E. Wilhelm,* Terence D. Murphy,* Robert W. Levis,* Erika Matunis,†
Nahathai Srivali,* Roger A. Hoskins‡ and Allan C. Spradling*,1
*Howard Hughes Medical Institute Research Laboratories, Department of Embryology, Carnegie Institution of Washington, Baltimore,Maryland 21218, †Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and
‡Department of Genome Biology, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720
Manuscript received September 18, 2006Accepted for publication December 18, 2006
ABSTRACT
Metazoan physiology depends on intricate patterns of gene expression that remain poorly known.Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancertrap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. Bysequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced greenfluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, wefound that 600–900 different genes are trapped in our collection. A core set of 244 lines trapped differentidentifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegiecollection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggestnew strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.
THE central challenge of postsequence genomics isto learn how an enhanced knowledge of genes,
transcripts, and proteins can be applied to betterunderstand the biology of multicellular organisms.Gaining an accurate picture of where and when meta-zoan genes are expressed remains a prerequisite formany such advances (Stathopoulos and Levine 2005).The discovery of distinctive, regulated programs ofgene expression at a fine scale has the potential to revealnew cell types and substructures that make up tissues andthe biological processes that govern their function.However, sensitive and widely applicable methods will berequired to detect and distinguish developmentallyprogrammed gene expression changes from thosecaused simply by cell cycling or environmental pertur-bation.
Several methods for analyzing gene expression withintissues are currently available. Particular cell types cansometimes be cultured in vitro into populations of usefulsize. However, isolated cells in artificial media frequentlybehave differently from cells in vivo interacting withprecisely positioned neighbors in three-dimensionalmicroenvironments. Another approach is to isolatetissue cells by flow sorting, microdissection, or lasercapture and then determine their expression profiles
in depth (reviewed in Espina et al. 2006). Visualizingpatterns of gene expression within the intact tissues oftransgenic organisms containing gene expression re-porters may be the most general method (Tomancak
et al. 2002). Epitope tagging, enhancer trapping, andgene trapping all have the added advantage that geneexpression can subsequently be observed in livingtissues, revealing dynamic processes that are largelybeyond the reach of methods based on fixed material(reviewed in Herschman 2003; Dirks and Tanke 2006).
Protein trapping is a variation of gene trapping inwhich endogenous genes are engineered to produceunder normal controls protein segments fused to areporter such as GFP. The great potential of thistechnology has been extensively documented in yeast,where large collections of strains that each trap adifferent gene have been generated using transposableelements (Ross-Macdonald et al. 1999) or by homol-ogous recombination (Huh et al. 2003). Extensive geneand protein trapping has also been carried out in cul-tured embryonic stem (ES) cells (Gossler et al. 1989;Friedrich and Soriano 1991), where fusions withmore than half of annotated mouse genes have beenrecovered (see Skarnes et al. 2004). However, relativelyfew of these ES cell lines have so far been used to gener-ate corresponding mouse strains where the versatilityand sensitivity of the method for analyzing tissue struc-ture can be tested.
1Corresponding author: Carnegie Institution, 3520 San Martin Dr.,Baltimore, MD 21218. E-mail: [email protected]
Genetics 175: 1505–1531 (March 2007)
Large-scale protein trap screens may also reveal newinformation about genome structure and function.Identifying in an unbiased manner locations through-out a genome where a coding exon can be expressedtests the accuracy and completeness of its annotation.Characterizing the splicing patterns that lead to normalor aberrant GFP expression tests the current catalog oftranscript isoforms generated by alternative splicing.Moreover, by recovering insertions in the same genethat splice differently and produce GFP with varyingefficiency, such a project might generate a data set usefulfor studying the determinants of splice site selection andtranscript stability.
Drosophila provides a favorable system for applyinggene traps to diverse developmental and genomicstudies. The genome sequence has been extensivelyannotated on the basis of experimental data (Misra
et al. 2002). Thousands of enhancer trap lines have beengenerated in large-scale transposon screens and culledof redundant strains by the gene disruption project (seeBellen et al. 2004). In contrast, producing Drosophilaprotein trap lines has remained difficult. Several hun-dred such lines were generated using a mobile GFP-containing exon flanked by both splice acceptor anddonor sites (Morin et al. 2001; Clyne et al. 2003).However, the process was highly inefficient, with as fewas 1 in 1500 progeny flies expressing GFP. Positive linesoften contained more than one insertion, preferentiallytagged a small number of hotspot loci, and tagged manysites not predicated to fuse the GFP exon in frame toany known coding region (Morin et al. 2001). Kelso
et al. (2004) found that the recovery of lines could beincreased by using an automated embryo sorter to selectGFP-positive embryos and established a website, Fly-Trap, to gather information on Drosophila protein traplines. Consequently, we initiated a large-scale proteintrap screen to increase gene coverage, test the genomeannotation, and address some of the remaining techni-cal difficulties in efficient line production.
Here we report the production of lines that trap 600–900 Drosophila genes, including 244 where one or moretrapped proteins can currently be identified. Using theDrosophila ovary as a test system we confirm that proteintrap lines reveal fine-scale details of developmentallyregulated protein expression, making them exception-ally valuable discovery tools for a wide range of studies.Finally, mapping RNA splicing patterns downstreamfrom .1200 insertions provides insight into how anadded exon affects splicing and suggests how the pro-duction of protein trap lines can be expanded to cover alarger fraction of the Drosophila proteome.
MATERIALS AND METHODS
Generation of P-element lines for protein trap screening:The P-element-based protein trap screens presented here uti-lized the pPGA, pPGB, and pPGC vectors described in Morin
et al. (2001). These elements carry a mini-white transgene inthe opposite orientation to an enhanced green gluorescentprotein (EGFP) exon, which is composed of EGFP sequence,without start or stop codons, flanked by splice acceptor anddonor sites from the Drosophila MHC locus. A, B, and C referto the position of the splice sites within the first and last codonsof the EGFP exon sequence. Previously used pPGA, pPGB, andpPGC third chromosome insertions (Morin et al. 2001) wereremobilized in the presence of balancer chromosomes. Newinsertions that mapped to the CyO balancer chromosome, didnot express EGFP, and exhibited remobilization rates off of theCyO balancer of at least 60% in single-pair mating assays wererecovered and used as starting stocks in the screen (see below).
piggyBac protein trap vectors: To make a shuttle vector forsubcloning the EGFP exon into different transposable ele-ments, the entire EGFP exons from the pPGA, pPGB, or pPGCplasmids were excised from the original P-element plasmids(kind gift of W. Chia) using EcoRI and PstI and subcloned intopBluescript (Stratagene, La Jolla, CA). These plasmids werethen cut with EcoRV and KpnI, end filled using Klenow, andreligated to themselves to create pBS-GFPA, pBS-GFPB, andpBS-GFPC. The resulting plasmids carry the EGFP exonsequence between a unique EcoRI site at the 59 end andunique PstI, SmaI, BamHI, and XbaI sites at the 39 end. Newtagging sequences can be inserted between the splice acceptorand donor sites of the exon using unique NcoI and XhoI sites.
Two different piggyBac protein trap vectors (Figure 1) wereconstructed using pBac{D. m. w1} (Handler and Harrell
1999) (kind gift of A. Handler). The pBac{D. m. w1} plasmidwas digested with ClaI to remove most of the mini-whitesequence and a linker containing HpaI, XhoI, and SpeI siteswas inserted in its place to form pBAC{DClaI}. To createpBAC{BglII-GFP}, the EGFP exons from pBS-GFPA, pBS-GFPB,and pBS-GFPC were subcloned into the BglII and MfeI sitesof pBAC{DClaI}. To create pBAC{HpaI-GFP}, EGFP exon se-quences were inserted between the MfeI and HpaI sites ofpBAC{DClaI}. An intronless yellow transgene from the yellow-BSX plasmid (Bellen et al. 2004) was then subcloned into theunique SpeI site of both pBAC{BglII-GFP} and pBAC{HpaI-GFP}to form either pBAC{BglII-GFP; y1}, which has the EGFP exonand yellow transgene oriented away from each other, orpBAC{HpaI-GFP; y1}, which has the EGFP exon and yellowtransgene pointing toward each other (Figure 1A). BothpBAC{BglII-GFP; y1} and pBAC{HpaI-GFP; y1} vectors carryingthe EGFP exon in the A frame were transformed into y w fliesusing the phspBac helper plasmid (Handler and Harrell
1999) (kind gift from A. Handler).We created stable genomic sources of the piggyBac tranpo-
sase using P-element transformation vectors. To place thepiggyBac transposase under control of the ubiquitin promoter,the piggyBac transposase ORF was excised from phspBac usingBamHI and DraI and ligated into the BamHI and SmaI sites ofthe pCasper3-Up2-RX poly(A) P-element vector (Ward et al.1998) (kind gift of R. Fehon), which carried a modifiedmultiple cloning site (kind gift of A. Hudson), to form pP{Ub-pBACtrans}. To make an inducible piggyBac transposase source,phspBac was digested with EcoRI and DraI and the fragmentcontaining both the Drosophila hsp70 promoter and piggyBactransposase ORF was ligated into the EcoRI and StuI sites ofpCasper4 to form pP{hsp70-pBACtrans}. These vectors wereused to transform y w flies, using standard P-element trans-formation techniques.
To test the activity of the piggyBac transposase transgenes,single-pair matings were set up using the pBAC{HpaI; y1}24.3insertion, which mapped to the X chromosome, and pP{Ub-pBACtrans} or pP{hsp70-pBACtrans} stocks. The pBAC{HpaI;y1}24.3 insertion was mobilized in males that were thenoutcrossed to y w females. Phenotypically yellow1 males in
1506 M. Buszczak et al.
the next generation were scored as new insertions. ThepP{hsp70-pBACtrans} was able to remobilize the pBAC{HpaI;y1}24.3 insert in 43% (n ¼ 30) of single-pair matings testedwhereas the pP{Ub-pBACtrans} was able to remobilize thepBAC{HpaI; y1}24.3 insert in 48% (n ¼ 21) of single-pairmatings tested. The pBAC{HpaI; y1}24.3 insertion was remobi-lized in the presence of a CyO balancer chromosome. Newinsertions that did not express EGFP and mapped to the CyOchromosome were used in the pBAC-based protein trap screen.
Generation of embryos with novel transpositions: Weisolated new EGFP-expressing P-element insertions using thefollowing genetic scheme:
F1 :yw
Y;
Sco
CyO pPGfw 1; GFP exong;1
13
yw
yw;
1
1;Ki; Pfry1t7 :2 ; D2 -3g99B
Ki; Pfry1t7 :2 ; D2 -3g99B
F2 :yw
Y;
1
CyO pPGfw 1; GFP exong;1
Ki; Pfry1t7 :2 ; D2 -3g99B3
yw
yw;
1
1;
1
1
Sort embryos: :
New pBAC insertions were generated through a similargenetic scheme:
F1 :yw
Y;
Sco
CyO pBACfy 1; GFP exong;1
13
yw
yw;
1
1;Pfw 1; pBACtransgPfw 1; pBACtransg
F2 :yw
Y;
1
CyO pBACfy 1; GFP exong;1
Pfw 1; pBACtransg3yw
yw;
1
1;
1
1
Sort embryos:
Hereafter, P-element and piggyBac element-based protein traplines were treated the same. For the F1 cross several hundredmales and females of the appropriate genotypes were matedin bottles to produce several thousand males in which theelements were mobilized for the F2 cross. These males werecrossed to 8000–10,000 virgin y w females in a population cage.These virgin females were obtained using a virgining stock thatcarried a heat-shock-inducible hid transgene on the Y chro-mosome (kind gift of R. Lehmann). Two separate overnightembryo collections from each population cage were screenedfor EGFP expression. We limited the number of times wescreened embryos from a particular cage to try to minimize thenumber of identical insertions recovered due to premeoiticinsertion events.
Embryo sorting and line establishment: We screened forEGFP expression in embryos using a COPAS Drosophilaembryo sorter (Union Biometrica). Embryos were dechorio-nated in 50% bleach for 2.5 min and washed extensively withwater. Dechorionated embryos were then washed into sortingsolution (0.53 PBS, 2% Tween-20). With the exception of thesorting solution, the COPAS sorter was used according to themanufacturer’s protocol, using the manufacturer’s solutions.The sorter and sample pressures of the COPAS machine andembryo density were maintained so that the COPAS sorterscreened 15–20 embryos/sec. The sorter used a 488, 514 nmmultiline argon laser. EGFP fluorescence was detected usingPMT1 set to 510 nm. Red fluorescence, used as a measure ofembryo autofluorescence, was detected using a second PMTset to 580 nm. Baseline values for each fluorescent axis were set
empirically using previously isolated fly strains that express lowlevels of EGFP and y w non-EGFP expressing embryos (Figure1). Approximately 250,000 embryos were sorted in five 50,000-embryo batches per day. Sorted embryos were collected andwashed in dH2O. All the embryos from a single batch wereplaced together in standard food vials. We estimate that�80%of the sorted embryos survived to adulthood. Sorted flies thatsurvived to adulthood and did not carry the Ki, P{ry1t7.2;D2-3}99B or P{w1; pBACtrans} chromosomes were individuallyoutcrossed to a y w stock. New lines that carried EGFP-expressing insertions that did not map to the starting CyOchromosome were maintained as stocks.
DNA sequencing, RT–thermal asymmetric interlaced PCRanalyses, and prediction of fusion potential: Genomic se-quences flanking either P-element or piggyBac protein trapinsertions were determined by members of the LawrenceBerkeley Lab group using an established protocol for sequenc-ing inverse PCR products from genomic DNA (Bellen et al.2004). Database software developed for the annotation of theDrosophila gene disruption project (Bellen et al. 2004) wasused to manage the sequence data. Once the insertion site of agiven protein trap line was determined, a FileMaker Pro data-base that contained information [version 3.2 of the Drosoph-ila genome annotation (Misra et al. 2002)] for all Drosophilatranscripts, exons and introns, and their reading frames wasused to predict which gene(s) and transcript(s) were beingtrapped by a given protein trap insertion.
We developed a reverse transcriptase coupled thermalasymmetric interlaced PCR (RT–TAIL) protocol largely onthe basis of methods used to determine T-DNA insertion sitesin Arabidopsis (Singer and Burke 2003). This methodallowed us to determine the mRNA sequence adjacent to theEGFP exon without using gene-specific primers. Total RNAwas isolated from 15 adult flies using an RNAqueous-96automated kit (catalog no. 1812; Ambion, Austin, TX). Thesamples were ground in 200 ml of sample buffer and spun for 5min at 14,000 rpm. The supernatant was placed in a 96-wellplate and 100 ml of 100% EtOH was mixed with each sample.The sample was transferred to the filter plate, washed, andthen treated with Dnase I (Ambion) for 15 min. Rebindingbuffer was added to each well of the filter plate, and the platewas washed extensively. The RNA was eluted off the filter andprecipitated with 7.5 m LiCl solution (Ambion). The resultingRNA pellet was washed with 75% EtOH and then retreatedwith Dnase I for 30 min at 37�. Dnase inactivation reagent(Ambion) was added to the samples. The RNA samples werespun and the supernatant was transferred to a new plate. Adetailed protocol is available upon request.
The following GFP-specific primers were used for RT–TAILPCR:
GFP-For1, 59-GGAGGACGGCAACATCCTGG-39;GFP-For2, 59-CAACGTCTATATCATGGCCG-39;GFP-For3, 59-AGACCCCAACGAGAAGCGCG-39;GFP-Rev1, 59-GTCGTGCTGCTTCATGTGGTCG-39;GFP-Rev2, 59-GACACGCTGAACTTGTGGCCG-39;GFP-Rev3, 59-AGCTCCTCGCCCTTGCTCACC-39 .
The arbitrary degenerate (AD) primers used in this studywere originally described by Singer and Burke (2003) but arelisted here for convenience:
AD3, 59-AGWGNAGWANCAWAGG-39;AD4, 59-STTGNTASTNCTNTGC-39;AD5, 59-NTCGASTWTSGWGTT-39;AD6, 59-WGTGNAGWANCANAGA-39.
A pool of the AD primers was mixed according to Singer
and Burke (2003).
Protein Trap Lines in Drosophila 1507
The first round of RT–TAIL PCR was set up in 96-well formatusing a one-step RT–PCR kit (QIAGEN, Valencia, CA). Forevery reaction 5 ml of total RNA was mixed with 10 ml 53 buffer,2 ml 10 mm dNTP solution, 1 ml GFP-For1 or -Rev1 primer, 12.5ml AD primer mix, 2 ml enzyme mix, and 17.5 ml of dH2O. Thereverse transcription reaction was carried out at 50� for 30 min.The sample was then heated to 95� for 15 min and then cycledfor primary TAIL–PCR according to Singer and Burke
(2003), using a MJ Research (Watertown, MA) thermal cycler.The secondary and tertiary TAIL–PCR reactions were carriedout according to Singer and Burke (2003), using GFP-For2 or-Rev2 primers and GFP-For3 or -Rev3 primers, respectively, andregular TAQ DNA polyermase (Roche, Indianapolis). ThePCR products of the tertiary reaction were treated with exoSAP(United States Biochemical, Cleveland) and sequenced usingGFP-For3 or -Rev3 primers.
The RT–TAIL PCR protocol using the three GFP-Forprimers, which amplified off of the 39 end of EGFP, consis-tently yielded better results than the same reaction using theRev primers. Therefore most of the RT–TAIL PCR data definesplicing products at the 39 end of the EGFP sequence. Toidentify sequence fusing to the 59 end of EGFP, we employed a59 RACE kit according to the manufacturer’s protocol (Am-bion), using the EGFP reverse primers listed above.
Analysis of protein expression in tissues: Samples weredissected in Grace’s medium, placed in 48-well plates outfittedwith a nylon mesh bottom, and fixed in 4% paraformaldehydebuffered in 13 PBS for 10 min at room temperature. The platewas washed extensively with PBT (13 PBS, 0.5% Triton X-100,0.3% BSA) and incubated overnight at 4� with rabbit anti-GFPantibody (Torrey Pines) (1:2000) in PBT. The samples werethen washed extensively with PBT and incubated with goatanti-rabbit Alexa488 (Molecular Probes, Eugene, OR) (1:400)for 4 hr at room temperature. The samples were then washedwith PBT, stained with 2 mg/ml of DAPI, and mounted inVectashield (Vector Laboratories, Burlingame, CA). Imageswere collected using a Leica SP2 confocal microscope.
RESULTS
Generating a large initial collection of tagged strainsexpressing EGFP: Our initial strategy was to generate amuch larger number of lines containing new proteintrap vector insertions than in previous screens and toinstitute additional technical improvements. Because oftheir proven utility, we used the same P-element-basedprotein trap vectors employed by Morin et al. (2001),but we also constructed a similar set of vectors withpiggyBac (Figure 1A). As described in materials and
methods, we set up crosses in small population cages tolimit the recovery of clusters, utilized dominant markersto remove the transposase source from all new lines, andidentified rare GFP-expressing embryos rapidly andsensitively using an automated embryo sorter (Figure1B). This protocol allowed us to screen .60 millionembryos over a period of 2.5 years, to identify .7500‘‘green’’ embryos, and to use each one to start anindividual culture (see Table 1). Ultimately, 7404 strainswere successfully established, maintained by selectionfor white1 eye color, and analyzed further as dia-grammed in Figure 1C.
The same scheme was used with both transposons;however, in practice the piggyBac vectors were not nearly
as efficient at generating EGFP-positive candidate linesas the P-element vectors (Table 1). P-element vectorstypically exhibited 70% mobilization and generated �1EGFP-expressing embryo per 1000 sorted. In compari-son, the piggyBac vectors displayed nearly 50% mobili-zation, but they yielded only 1 EGFP-expressing embryoper 50,000 sorted. Thus, the piggyBac vectors wereslightly less efficient at mobilization, but drastically lessefficient at generating EGFP-positive lines upon in-sertion. Consequently, we soon abandoned attempts togenerate large numbers of piggyBac protein trap inser-tions (Table 1), but continued to characterize the lineswe did recover to learn if they would shed any light onthe lower frequency of trapping observed.
Localizing insertions on the annotated genome: Toidentify candidate proteins that may have been fusedwithin individual lines, we determined the genomicDNA sequence flanking the insertion(s) in collabora-tion with the Berkeley Drosophila Genome Project(BDGP) gene disruption project (materials and
methods). In most cases, the sequences from boththe 59 and 39 vector end junctions mapped by BLASTanalysis to a unique insertion site within the Drosophilagenome sequence. Lines for which the sequencingreaction failed, the sequence matched repetitive DNA,or the 59 and 39 sequences differed (indicating that twoor more insertions were present in the stock) wererecycled back into the starting pool, and frequently aunique single insert was eventually identified. Alto-gether the insertions in 1375 C frame, 3172 B frame,and 1009 A frame P-element and 164 piggyBac A framelines were localized to unique genomic sites.
Knowing the genomic location of an insertionallowed us to predict which transcripts would incorpo-rate the EGFP exon and whether they would undergosplicing and translation into a functional fusion protein.First, we removed �1550 duplicate lines derived frompremeiotic clusters that were identified because theybore insertions identical in position and orientation tothose in one or more sibling lines. Of the 4170 in-dependent lines remaining, 2149 (52%) were associatedwith a gene correctly oriented for possible fusion (i.e.,located between �500 and the 39 end). We also clas-sified the ways an insert can be located relative to itsclosest annotated transcript into general categories asdiagrammed in Figure 1D and classified all the lines(Table 2).
Expression of a fusion protein is expected when theGFP exon resides between two coding exons within anintron of matching reading frame (class 1A). Such inser-tions made up only 23% of the total localized insertionsand defined 192 different genes (Table 4). Forty percentof insertions were close to an annotated gene but werenot predicted to express the EGFP exon (classes 2–4),while 37% of the lines were not located within 0.5 kb of acorrectly oriented gene. EGFP production from theselines might be explained by the use of unannotated
1508 M. Buszczak et al.
Figure 1.—Generation and classification of protein trap vector insertions. (A) Schematic of protein trap vectors (after Morin
et al. 2001). (B) Sample output from automated sorting of Drosophila embryos mobilized from site not expressing GFP. RareGFP1 embryos (red circles) registering above a threshold value are diverted by the machine and later used to start individualcultures. (C) Scheme for characterization of putative protein trap lines (see text). (D) Classification of the general types ofrelationships between transposon inserts and the local genome annotation. Classes 1–4 consist of insertions in the appropriateorientation located within a codon intron (class 1), a noncoding transcribed region (class 2), an upstream genomic region (class3), or an exon (class 4). For each class, the insert was either of the appropriate frame (subclass A) or of nonappropriate frame
(Continued)
Protein Trap Lines in Drosophila 1509
genic elements, by noncanonical splicing events, or bythe presence of a second insertion at a canonical site.
The Drosophila genome annotation is highly sup-ported by experimental evidence (Misra et al. 2002);hence the frequency of these discrepancies was sur-prising, but a similar outcome has been reported inprevious protein trap screens using both yeast (Ross-Macdonald et al. 1999) and Drosophila (Morin et al.2001). Some protein-coding genes may have beenmissed within cDNA libraries (Hild et al. 2003), and asubstantial number of genes may contain unannotatedfar-upstream promoters and alternative translation startsites. There might be a large class of RNA genes thathave escaped detection but that can drive expression ofEGFP using cryptic start sites. Alternatively, the highselective pressure used to isolate EGFP1 strains mayhave led to the recovery of rare events in which thenormal gene or transcript structure has changed.
Analysis of fusion transcripts: We sequenced por-tions of the fusion transcripts to address how EGFPexpression arises in lines of various classes. A limitednumber of 59 RNA sequences were obtained by 59 RACEanalysis or by TAIL–PCR (see materials and methods).As expected, several lines in class 1A were found toinitiate in normal exons upstream from the insertionsite. However, we discovered several CB lines that
spliced into the EGFP exon from the 59 P-element se-quences of the vector. The P-element promoter is highlyefficient at enhancer trapping, and the entire first exonof the transposase gene is present in the vector alongwith the start of intron 1, which is in the frame com-patible with CB lines. These lines were associated withnuclear localized EGFP, possibly due to fusion of thefirst exon of P transposase with EGFP. Further evidenceof enhancer trapping was observed in the analysis of lineCA07138. The EGFP RNA was fused to sequences, in-cluding an in-frame ATG start codon, derived by tran-scription and splicing from the noncoding strand of themini-white transgene carried in the P-element vector(Figure 1E). These observations suggest that EGFP ex-pression in a significant number of the lines depends ontranscripts initiated from within the transposon itself byenhancer trapping rather than on EGFP exon additionby splicing into exogenous transcripts.
Analysis of downstream transcript sequences: Togain additional information, we analyzed the sequencesdownstream from the EGFP exon from many lines in thecollection by carrying out RT–TAIL–PCR in a 96-wellformat (materials and methods). 39 sequences up to700 bp in length and defining the location of one tosix downstream exons were obtained for .1200 lines(Table 3). The pattern of downstream splicing allowedproductive fusions to be identified and indicated linesthat splice out-of-frame and likely become subject tononsense-mediated decay (NMD) (Vasudevan andPeltz 2003). Fusions within lines of classes 2–4 often
(subclass B) to fuse to the protein if splicing continued to the next annotated exon splice acceptor site. Class 5 consists of trans-posons inserted .0.5 kb from a correctly oriented annotated gene. (E) The structure of cryptic transcripts initiated within theDrosophila mini-white marker gene that contain an ATG codon and splice in frame to EGFP, thereby allowing expression inde-pendent of an endogenous transcript in some lines. (F) Western blot analysis of Dlg1 and eIF-4E protein production in controlanimals (y w, CC00380) and insertion lines predicted to trap Dlg1 (CC01936) or eIF-4E (CC00392, CC00375, and CC01492). (G)Abnormal nuclear accumulation of CG15015-EGFP in line CC01311 whose insertion lies within the FHC domain (left). Tissueculture cells expressing N-terminal or C-terminal fusions are found in the cytoplasm (center and right).
TABLE 1
Project summary
Type No. %
Total setup 7404 100CA 1344 18CB 4000 54CC 1870 25piggyBac A 190 3Aligned sequence 5720 77Clusters 1550 21Independent aligned 4170 56Gene hits 2149 29Spacing hits 2093 28Different genes 1154 16Protein traps 244Enhancer traps 256Novel gene/exon 50Unclassified 300Balanced stocks 878 12
TABLE 2
Line types
Lines
Type Code N %
Coding intron, in frame 1A 1337 23Coding intron, out of frame 1B 173 3Noncoding intron, in frame 2A 29 1Noncoding intron, out of frame 2B 429 81–500 bp upstream, in frame 3A 56 11–500 bp upstream, out of frame 3B 756 13Exon 4 804 14.500 bp to next oriented gene 5 2093 37Total 5677 100
See class definitions in Figure 1D.
1510 M. Buszczak et al.
were predicted to encode a ‘‘linker peptide,’’ whichmight or might not include a stop codon, derived fromthe translation of a small segment of upstream nucleo-tides. The RNA analysis also revealed the presence of asecond insertion in 14% of type 1A lines, but between 24and 50% of the other classes. The second insertionsfound within class 2–5 lines were often valid protein trapalleles (class 1A) and were frequently the true source ofthe lines’ EGFP production. This information allowedus to identify additional candidate fusions (Table 4), tocorrect many initial line classifications, and to moreaccurately estimate the total number of trapped genes(Table 1: 600–900). By the time lines were selectedand balanced, secondary insertions or damage did notcontribute substantially to the phenotypes reported inTable 4. Tests estimated the frequency of backgroundlethal mutations among balanced, saved lines at 7–21%,similar to the best transposon screens (Spradling et al.1999).
Novel splicing suggests new genes and exons: Wecompared the splicing observed downstream from theinserted exons with that of the genome annotation(Misra et al. 2002) to identify new Drosophila gene andtranscript isoform candidates. To identify new candi-date genes, we focused on lines inserted .0.5 kb froman appropriately oriented known gene and for whichRNA sequence data were also available. In 114 of these205 lines, the RNA sequence coincided with the positionand orientation of the insertion and therefore indicatedthe splicing pattern downstream of the single EGFPexon. Most of the lines spliced to one or more novelexons. At least 8 probably correspond to unannotatedgenes because they match previous gene predictions(Hild et al. 2003) or are supported by EST data (seeTable 4). Most of the remaining exons do not predictproteins with homologs in other species and representeither aberrant splicing events or novel or untranslatedexons.
Similar analysis of 297 lines with intron insertionsallowed us to test for novel exons and transcriptisoforms. We examined 443 splicing events and identi-fied a total of 35 (7.9%) that did not correspond tocurrent gene models (Misra et al. 2002). Since at least
some of these differences probably resulted from aber-rant splicing induced by the insertions, this represents amaximum estimate of the fraction of unannotatedgenomic exons and emphasizes the high accuracy andcompleteness of current Drosophila gene models, atleast for abundant transcript forms. Often, the RNAdata indicated which isoform among several predictedto fuse in frame is likely to predominate in ovariantissue. For example, we could determine that lineCA06613 in ovarian tissue predominantly fuses theSu(Tpl) gene rather than Mi-2, in whose transcriptionunit it also lies in frame.
The nature of the noncanonical splices observed wasinteresting. The most common events (21/35) were forinsertions in large introns to splice to a novel exon(s)prior to joining the predicted downstream exon. Somesimply appear to define alternative isoforms that skipexons or utilize different exon combinations not pre-viously documented. Some of these events may havebeen induced by the abnormal position of the EGFPexon within the primary transcript. However, severallines appear to define alternative isoforms because theyutilize different combinations of known exons in nopreviously documented transcript isoforms. Three linesutilized 59 start sites for exons that differed by 6, 21, or27 bp from the annotated exon. The CC01473 transcriptreads through an annotated exon into the adjacentintron and probably defines a novel alternate transcript39 end. Although we consider it likely that many of thesedifferences reflect endogenous Drosophila gene ex-pression, all of the candidate novel genes and transcriptisoforms require independent confirmation in strainslacking protein trap insertions. Such tests were beyondthe scope of our project.
Protein trap insertions likely vary in the fraction of theendogenous protein that is tagged with EGFP for avariety of reasons. First, in many lines only some of themultiple-transcript isoforms contributing to proteinproduction are tagged by the insertion and fused inframe. Second, the splicing efficiency of the EGFP exonmight vary due to its surrounding genomic context. Toinvestigate this issue, we analyzed the protein productsof tagged genes by Western blotting. The tagged pro-teins were easily distinguished from their wild-typecounterparts on the basis of size and by probing withprotein-specific and anti-EGFP antibodies (Figure 1F).In line CC01936 all three isoforms are predicted toincorporate the EGFP exon in frame, and nearly all ofthe ovarian Dlg1 protein incorporated EGFP as in-dicated by its mobility. A similar result was reportedpreviously in the case of line CB02119 (Buszczak andSpradling 2006), where the precursors of five of sixannotated transcripts are predicted to contain theinsertion, although only two fuse in frame. In contrast,only �50% of the ovarian wild-type eIF-4E protein istagged with EGFP (Figure 1F) despite the fact that six ofseven annotated eIF-4E transcripts initiate upstream
TABLE 3
RNA analysis
Type SuccessfulConfirmed
DNASecondinsert
%confirmed
CA 316 224 50 82CB 328 166 75 69CC 572 165 72 70Piggy A 13
Totals 1229 555 197 74
Protein Trap Lines in Drosophila 1511
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Protein Trap Lines in Drosophila 1517
from the insertion site. These examples suggest that theEGFP incorporation level varies between genes in largepart due to the tagging of a subset of gene isoforms thatthemselves display differing expression levels.
In contrast, we found little evidence of short-rangecontext effects. Less than twofold variation in proteinexpression as measured by Western blotting with anti-EGFP antibodies was observed between lines withinsertions at sites within the same intron (N. Srivali
and A. Spradling, unpublished data). However, theseexperiments did reveal that insertions of the piggyBac-based vector consistently produced less EGFP proteinthan lines with the corresponding P-element-basedvector that were inserted in the same intron (N. Srivali
and A. Spradling, unpublished data). This suggeststhat some aspect of the structure of the piggyBac vectorused compromised splicing efficiency.
Identification of protein trap and enhancer trap corecollections: To help identify a core set of valid gene traplines we examined the EGFP expression patterns ofmany nonredundant lines in both the adult ovary andlarval salivary gland. There was a strong correlationbetween insert location and the nature of the stainingpatterns observed. More than 95% of lines in class 1A,the in-frame fusions, produced patterned EGFP expres-sion above background in at least some ovarian cells orin the salivary gland. In contrast, a much smaller, but stillsignificant, fraction of lines in classes 2–5 also expressedEGFP in a regulated manner. Combining informationon insert location, genome annotation, RNA transcriptsequence, and EGFP pattern, we identified a set of 244lines predicted to produce fusions between EGFP and431 protein isoforms of 233 distinct genes (Table 4).These new protein trap lines express EGFP in a widevariety of cellular compartments under diverse devel-opmental controls (see Figures 2 and 3).
A second major class of lines in the collection showedthe properties expected of EGFP enhancer traps (Ta-ble 5). These CB lines were susceptible to enhancertrapping from the P-element promoter, were locatedmostly upstream of the annotated start site, expressedEGFP in nuclei, and the RNA analysis, if available, didnot indicate fusion in frame downstream. The expres-sion patterns of such insertions in well-characterizedgenes supported this interpretation. For example, lineCB02030 in ptc showed strong expression in the innersheath cells of the germarium (Forbes et al. 1996), whileline CB04353 in Dad strongly expressed in the germlinestem cells and immediately downstream germ cells (Kai
and Spradling 2003; Casanueva and Ferguson 2004).The characterization of a significant number of lines
in the collection remains incomplete (Table 1). Many ofthese contain insertions located .0.5 kb from an ap-propriately oriented annotated gene but where RNAsequence data were not obtained. Others are insertedwithin genes at locations not predicted to generateprotein fusions or gene traps. Some of these lines
TA
BL
E4
(Co
nti
nu
ed)
Gen
eL
ine
Site
aC
hr
Stra
nd
Ph
eno
typ
eT
1In
sert
Met
Sto
pT
2In
sert
Met
Sto
pT
3In
sert
Met
Sto
pT
4In
sert
Met
Sto
pT
ype
CB
0265
812
5220
672R
�h
v5B
CC
0167
012
9448
373R
1h
v5B
CC
0193
221
8568
623R
�h
v5B
CA
0743
656
4435
82R
�sl
5BC
B03
064
8536
402
2L1
hv
5BC
C00
523
2280
6145
3R�
hv
5B
aA
nn
ota
tio
nre
leas
e5.
0.
1518 M. Buszczak et al.
express EGFP in the ovary, and we cannot rule out thatothers express transcripts in other tissues. On the basisof the processing of previous lines in these same classesthis suggests that a significant number of new enhancertraps and a handful of new protein traps could be sortedout from a larger number of lines with secondary inser-tions in already trapped genes. Consequently, thenumber of different genes trapped in the collectionis likely to increase beyond the 600 or so currentlycharacterized.
Even when a protein is tagged in frame, the insertionof the EGFP sequence is expected to disrupt its normalstructure and localization some fraction of the time. Forexample, line CC01311 traps CG15015, the Drosophilahomolog of mammalian Cip4, a modular proteinthat interacts with Cdc42 and helps to regulate theactin cytoskeleton (Aspenstrom 1997). The CC01311P element is inserted between the first two coding exonsand thus disrupts the FES/Cip4 domain of CG15015(Figure 1G). The protein trap fusion product localizesto the nucleolus while transgenes of CG15015 tagged at
either the very N or C termini localize to the cytoplasmwhen expressed in S2 cells. We observed that 3 otherprotein trap fusion products of 107 analyzed accumu-lated in the nucleus when they were expected to residein the cytoplasm.
Diverse behavior of tagged proteins: The 244 iden-tified protein trap lines of the core collection exhibitextremely diverse patterns of EGFP expression, suggest-ing that proteins occupying a wide range of cellularcompartments can be tagged in vivo. We observed manylines with EGFP fluorescence in the cytoplasm (Figure2A) or nucleus (Figure 2B) as expected. Localization tointracellular membranous structures was also com-monly seen, as illustrated by a trap in the ER structuralcomponent Reticulon-1 (Figure 2D). Gene trapping ofsecretory proteins is thought to be inefficient due toretention of the fusion proteins in the ER where theactivity of the fusion gene may be affected (Skarnes
et al. 1995). The full-length protein traps we constructedcould label secreted proteins, as indicated by theextracellular localization of EGFP in CA06735, a fusion
Figure 2.—Protein traps for the study of pro-tein subcellular localization. Patterns of subcellu-lar localization of EGFP expressed from thefollowing lines that trap the indicated genes wereobserved: (A) cytoplasmic, CA06607 (CG17342);(B) nuclear, BA00164 (dom); (C) enhancer trapnuclear, CB04353 (Dad) in stem cells and earlycystocytes; (D) endoplasmic reticulum,CA06523 (Rtnl1); (E) extracellular, CA06735 (ca-thepsin K); (F) membrane, CA07474 (Picot); (G)apical, CC01941 (Baz); (H) chromatin, CA07249(stwl); (I) nuclear membrane, CA07301(Fs(2)Ket); (J) lipid droplets, CA07051 (Lsd-2);(K) novel structure, CA07332 (CG6854); (L)novel structure, CC01326 (polo).
Protein Trap Lines in Drosophila 1519
Figure 3.—Protein traps for the study of developmental regulation during oogenesis. The expression in the ovary of variousprotein trap lines is shown to illustrate how they can be used to associate genes with developmental processes. (A) Schematic of anovariole tip. The terminal filament (TF), cap cells (CpC), germline stem cells (GSC), cystoblast (CB), and escort cells (ES) areillustrated. (B–H) Cell type identification. (B) Terminal filament, CB02069 (CG14207); (C) cap cells, CB03410 (CG1600); (D)escort cells, CC01359 (fax); (E) follicle cells, CC06135 (CG12785); (F) outer border cells and posterior follicle cells, CB02349(NK7.1); (G) oocyte nucleus equals the germinal vesicle (arrow), CB04219 (CG13776); (H) novel sheath cell type, CC01646(CG12920). (I–L) Analyzing developmental processes. (I) Novel structure in center of midstage follicle, CC00523 (Msn); (J) fu-some, CC01436 (Sec61); (K) germline and somatic ring canals, CA07004 (Vsg); (L) chorion gene amplification, CB04400 (Orc2).(M–P) Localization of proteins in the oocyte. (M) Posterior pole, CC01442 (EIF-4E); (N) posterior pole, CA06517 (Tral); (O)posterior pole, CC00236 (CG32423); (P) anterior pole, CA07529 (CG6151). (Q–T) Developmental regulation of gene expressionin early germ cells. (Q) Control with little change, CC01961 (Actn); (R) GSC/CB enriched, CC06238 (CG11963); (S) GSC andearly cyst enriched, CC01915 (Eff); (T) GSC and forming cyst enriched, CC01442 (EIF-4E).
1520 M. Buszczak et al.
TA
BL
E5
En
han
cer
trap
alle
les
Gen
eL
ine
Site
aC
hr
Stra
nd
Ph
eno
typ
eT
1In
sert
Met
Sto
pT
2In
sert
Met
Sto
pT
3In
sert
Met
Sto
pT
4In
sert
Met
Sto
pT
ype
1.28
CB
0449
223
8942
52R
1h
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A0.
51
13
4EH
PC
B05
417
1993
4636
3R�
hv
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11
44
abo
/l(
2)06
225
CB
0327
210
9753
072L
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A0.
51
23
Ad
f1C
B02
276
2550
455
2R�
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hal
RA
2.5
14
1A
dk3
/C
G46
74C
B04
307
6715
035
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hv
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0.5
22
RA
0.5
22
3ao
pC
B02
762
2178
398
2L�
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hal
RB
12
44
AT
PC
L/
CG
8370
CB
0442
711
8887
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A0.
52
93
bic
CB
0385
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5772
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52
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13
bn
lC
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854
1566
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0.5
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RB
0.5
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0207
034
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bo
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586
5416
706
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11
24
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0266
514
9654
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btn
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0350
118
4140
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A0.
51
43
Cct
1C
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171
1546
105
3L1
hv
RA
12
44
cdi
CB
0512
914
9199
503R
1h
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A1
28
22
CG
1022
5/T
bp
-1C
B02
343
1957
8503
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hv
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0.5
13
3C
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CB
0356
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3308
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1.5
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1.5
39
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399
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51
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CG
1044
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265
1616
1990
2R�
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hal
RA
11
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3942
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11
63
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0C
B02
351
1364
8912
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hv
RA
12
45
CG
1104
2C
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720
9046
478
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hv
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0.5
11
3C
G11
16C
B03
511
1045
469
3R�
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11
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11
54
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1138
2C
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1103
926
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RB
0.5
11
3C
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526
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0222
833
2588
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51
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1153
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0.5
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0.5
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2767
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hal
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12
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5700
359
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hv
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0.5
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cbx
CB
0509
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0.5
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0223
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1334
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24
(con
tin
ued
)
Protein Trap Lines in Drosophila 1521
TA
BL
E5
(Co
nti
nu
ed)
Gen
eL
ine
Site
aC
hr
Stra
nd
Ph
eno
typ
eT
1In
sert
Met
Sto
pT
2In
sert
Met
Sto
pT
3In
sert
Met
Sto
pT
4In
sert
Met
Sto
pT
ype
CG
1454
2C
B02
373
2187
8549
3R�
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hal
RA
0.5
16
3C
G14
709
CB
0439
773
9488
63R
1h
vR
A0.
51
83
CG
1621
CB
0204
233
8060
22R
�h
vR
A0.
51
23
CG
1667
CB
0547
757
3128
02R
1Se
mil
eth
alR
A2
15
4C
G16
708
CB
0438
811
9353
33R
�h
vR
A1.
52
42
CG
1681
7C
B02
934
5503
678
3R1
hv
RA
11
44
CG
1697
1C
B03
898
2241
063L
1h
vR
B1
23
RC
12
3R
D1
23
4C
G17
002/
vim
arC
B05
094
2873
307
2R1
hv
RB
11
54
CG
1709
0C
B03
116
5442
483L
1h
vR
B1.
52
102
CG
1709
0C
B02
270
5442
853L
�h
vR
A1.
51
25
CG
1732
3C
B02
962
1882
3565
2L1
hv
RA
0.5
25
3C
G17
836
CB
0526
314
7495
493R
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B3.
53
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A3.
53
5R
C2.
53
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D0.
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1910
/l(
3)s1
921
CB
0370
227
5732
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2051
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0362
516
1485
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RA
0.5
13
RC
0.5
13
4C
G21
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020
1075
1743
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0370
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739
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RA
22
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CG
3147
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1500
6356
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(con
tin
ued
)
1522 M. Buszczak et al.
TA
BL
E5
(Co
nti
nu
ed)
Gen
eL
ine
Site
aC
hr
Stra
nd
Ph
eno
typ
eT
1In
sert
Met
Sto
pT
2In
sert
Met
Sto
pT
3In
sert
Met
Sto
pT
4In
sert
Met
Sto
pT
ype
CG
3398
2C
B04
965
1552
7849
3L1
hv
RA
11
13
CG
3409
CB
0441
226
0232
72R
�h
vR
A1.
53
72
CG
3411
0C
B04
263
2101
0670
3R1
Let
hal
RC
2.5
19
1C
G34
28C
B02
131
9480
857
3L�
hv
RA
11
34
CG
3654
/U
ch-L
3C
B04
163
9470
143
3L�
hv
RA
0.5
14
3C
G40
91C
B02
284
1958
9843
2R�
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hal
RA
12
3R
C1
34
4C
G43
00C
B04
249
1227
0328
3L�
hv
RA
11
4R
B1
14
4C
G45
70C
B02
124
6682
617
3R1
hv
RA
11
14
CG
4612
CB
0533
120
4136
412R
�h
vR
A0.
52
33
CG
5381
CB
0461
610
3219
492L
1h
vR
A0.
52
73
CG
5543
CB
0485
419
6967
642R
1h
vR
A0.
51
13
CG
5548
CB
0566
714
9578
72X
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52
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CG
5677
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0205
420
0557
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A1
11
4C
G60
14C
B04
785
2139
0388
3L1
hv
RA
1.5
37
2C
G62
18C
B03
213
1116
8745
3R1
hv
RA
12
54
CG
6311
/N
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4C
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145
1752
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3L1
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11
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CG
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0383
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(con
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1526 M. Buszczak et al.
with CG8947, a Drosophila cathepsin K homolog(Figure 2E), and the membrane location of a trap inPicot, a phosphate symporter (Figure 2F). Proteins thatare apically localized in polarized epithelia such asovarian follicle cells were easily visualized, as observedfor Bazooka (Par3) (Figure 2G). Subcompartmental-ized nuclear proteins were also readily apparent. Forexample, a trap of the Stonewall (Stwl) HMG-relatedprotein involved in germ cell chromatin organization(Clark and McKearin 1996) labeled nurse cell nucleiand the oocyte nucleus (inset) differently (Figure 2H).Fs(2)Ket, a protein involved in nuclear import, waslocalized to the nuclear periphery (Figure 2I). In somecases, cell-specific cellular compartments were labeled,such as in CA07051, which traps Lsd-2 and exhibitedEGFP localization to lipid droplets that arise in late-stage nurse cells (Figure 2J).
These studies provide a high-resolution view ofknown protein locations in living cells and also identifymany proteins that were not previously known to residewithin these compartments. In addition, the value ofprotein trapping as a discovery tool was illustrated by thefact that we observed new patterns of localization as well.For example, the HDAC4 protein, fused by the CA07134trap, labeled a small body often found in only one nursecell within an egg chamber (Figure 2K). A spindle-likestructure in young nurse cells was labeled with a proteintrap in the polo gene encoding a mitotic kinase (Figure2L). Antibodies specific for the trapped protein can beused to isolate and further investigate the proteinspresent in these novel structures.
Analysis of developmental regulation—ovarian cells:All the lines in the core collection were characterized onthe basis of their patterns of expression in germ cellsand follicle cells during oogenesis. These experimentsidentified lines expressing in the major classes ofsomatic cells, including terminal filament cells (Figure3B), cap cells (Figure 3C), escort cells (Figure 3D),profollicle cells (Figure 3E), and border and posteriorcells (Figure 3F). Other lines expressed in germ cells ofvarious ages, including some that were highly enrichedin the oocyte nucleus (germinal vesicle) (Figure 3G). Asin the case of subcellular compartments, these studiesdocumented patterns of developmental expression formany genes that were not previously known. Thesegenes become attractive candidates for study of theirfunction in the corresponding processes.
Strikingly, the collection also revealed the likelyexistence of new cell types and novel biological pro-cesses previously unrecognized despite many years ofstudy of ovarian biology. Line CC01646 traps theCG12920 protein and is expressed in a small subset ofovarian sheath cells that likely represent a novel cell type(Figure 3H). In line CC00523 we observed accumula-tion of Msn-EGFP preferentially at the center of mid-stage growing follicles (Figure 3I). It was not previouslyknown that this region was the site of unique protein
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Protein Trap Lines in Drosophila 1527
accumulation. Msn encodes a protein involved in Junkinase signaling, suggesting that a special intercellularjunction may assemble in this region to structurallyorganize the nurse cells. We observed a similar expres-sion program (not shown) for the line CB03040 thatfuses the Pli gene, encoding a protein associated withthe NF-kB signaling response.
Developmentally specific subcellular structures, in-cluding the fusome (Figure 3J, arrow) and both somaticand germline ring canals (Figure 3K), were also labeledby rare lines. A general and extremely useful applicationof the collection is to identify new proteins that areassociated with such structures and analyze the effect ofmutations. For example, the preferential accumulationof Sec61 in the fusome observed here has been validatedin recent studies (Snapp et al. 2004). Proof of principleexperiments of this type that focus on the fusome will bedescribed elsewhere.
Another valuable capacity of protein trap lines is theability to follow important developmental processes athigh resolution and in living cells. During oogenesis, atleast four major clusters of chorion genes undergospecific gene amplification in stage 10B follicles, aprocess that can be visualized as small ‘‘amplificationdots’’ of BrdU incorporation (Calvi et al. 1998). Theamplifying genes specifically contain substantial amo-unts of replication initiation proteins such as Orc2 atthis time, whereas normally Orc2 is found throughoutthe cell nuclei (Royzman et al. 1999). A protein trap linein Orc2 allows the amplifying loci to be directly visu-alized (Figure 3L). Inspection shows that the dots arenot present in preamplification stage follicle cells butstrongly label amplifying gene loci at stage 10B (Figure3L, arrow).
The Drosophila oocyte represents an importantmodel system for studying RNA and protein localiza-tion. Several biochemical and genetic studies haveidentified proteins enriched at either the anterior orthe posterior pole of the oocyte (Lasko 2003; Wilhelm
and Smibert 2005). Protein traps in genes identified inthese studies, including EIF-4E (Figure 3M) and Tral(Figure 3N), faithfully recapitulate the localization pat-terns of their endogenous counterparts to the posteriorpole of the oocyte (Wilhelm et al. 2003, 2005). Severalother proteins tagged in the collection display posteriorlocalization patterns including a trap in CG32423(Figure 3O), a largely uncharacterized RNA-bindingprotein. In addition, a trap in CG6151 appears to beenriched at the anterior of the oocyte (Figure 3P).While future work will clarify the role of these proteinsin oocyte patterning, these examples show that proteintrapping can complement other approaches and beused to identify new components of localized RNPcomplexes within the cells.
Protein traps provide unique opportunities to analyzegene regulation during development in populations ofcells that are difficult to isolate and in those that are
sensitive to loss of cellular context. We illustrate thepotential of this approach using the regulation of germcell development within and just downstream from thegermline stem cells (GSCs). Many protein trap lines,including CC01961 in Actn, showed uniform expressionin GSCs, CBs, and developing germline cysts (Figure3Q). However, it was possible to find other exampleswhere expression levels between GSCs and early germcells differed from those in other germ cells within thegermarium. One of the most striking examples was lineCC06238 that traps the putative Drosophila succinateCoA ligase gene. Expression was stronger in stem cells(and sometimes early cystoblasts) than in later germcells as illustrated in Figure 3R. Several other genes weredownregulated shortly after GSC division, including effete(Figure 3S), encoding the UbcD1 ubiquitin-conjugatingenzyme that has been shown toaffect germline cyst forma-tion (Lilly et al. 2000). Another line whose EGFP expres-sion was downregulated slightly later, at the completion ofcyst formation, trapped the Drosophila eIF-4E gene(Figure 3T). Downregulation of a related gene CG8023was previously observed at a slightly earlier time, duringcyst divisions (Kai et al. 2005).
Studies on the limitations of current protein trapmethods: We also tested the sensitivity of the approachused here to detect Drosophila genes by looking at theexpression of the lines in the core collection in germlinestem cells. First, although lines were selected on thebasis of expression in embryos, we found ovarianexpression above background in .90% of lines in thecore collection. However, this does not address whethermany other genes exist that were fused but expressedEGFP at levels too low to detect in either tissue. Analysisof germline stem cell RNA by hybridization to Affymet-trix arrays detected transcripts from �6500 Drosophilagenes over an �1000-fold dynamic range (Kai et al.2005). Although translational regulation and differen-tial protein stability, not to mention differences in stain-ing sensitivity between different preparations, would beexpected to introduce potential variation, we werecurious whether protein trap lines could detect stemcell gene transcripts across the full range of expressionlevels.
We observed a strong correspondence between thesetwo measures of stem cell gene expression (Figure 4, A–E). Lines with very strong EGFP expression tended tohave RNA levels at least 10-fold higher than lines withabove background but relatively weak expression. Theselines in turn had signals higher than most lines scored asbelow the level of detection on arrays. The correlationwas not perfect; for example, some lines showed moreEGFP expression in stem cells than might be expectedfrom the microarray study (Figure 4F). The existence ofsuch lines was not unexpected, because some lines likelystill carry second insertions, and the microarray usedwas based on release 1 gene models. Overall, we coulddetect EGFP above background in GSCs from nearly all
1528 M. Buszczak et al.
lines whose levels of mRNA are called as ‘‘present’’ onAffymetrix arrays (Kai et al. 2005). This suggests thatprotein trap lines are not limited to a relatively smallnumber of highly expressed genes, but can be used tofollow a large fraction of gene activity.
DISCUSSION
The Carnegie protein trap collection—a versatileresearch tool: These experiments significantly expandthe number of protein trap lines available for studies ofgene expression within a complex multicellular animal(also see accompanying article by Quinones-Coello
et al. 2007, this issue). Our initial characterization ofthese lines extends previous documentation that thebehavior of the EGFP-tagged protein often correspondsto the behavior of the protein to which it is fused.Moreover, we demonstrate that collections such as oursare exceptionally useful as tools of gene discovery.Candidate genes can be selected on the basis of thedevelopmental expression, subcellular localization, ordynamic behavior of particular protein isoforms. Thesame line can subsequently be used to purify the proteinand its associated complexes and to create deletions forfurther genetic analysis. The Carnegie collection isavailable for research use from the Carnegie Institution.Information is available at http://www.ciwemb.edu/resources/proteintrapcollection.html and at http://flytrap.med.yale.edu/.
Subcellular distribution of protein location: Previ-ously, gene and protein trapping in yeast has been usedto estimate the fraction of proteins that are localizedto various cellular compartments (Ross-Macdonald
et al. 1999; Kumar et al. 2002; Huh et al. 2003). Morethan half of all proteins showed a simple localizationto the cytoplasm or nucleus. Other subcellular struc-tures labeled by tagged proteins in yeast included the
plasma membrane, the ER, mitochondria, the lysosome,and the perixosome. We obtained similar results. Thedistribution patterns of EGFP-tagged proteins withinDrosophila ovarian cells generally matched those ofyeast and most known structures within egg chambershave now been labeled with at least one protein trap line(this study; Morin et al. 2001; Clyne et al. 2003). More-over, the large size of ovarian cells often allowed us todistinguish the fine structure of several subcellular com-partments labeled by EGFP fusion proteins generatedin this screen.
Developmental regulation of protein expression:Despite the fact that only one tissue was examinedclosely, a large number of proteins in the core collectionwere expressed and many were developmentally regu-lated. A diverse array of cell types within the germariumincluding the terminal filament, cap cells, escort cells,germline stem cells, and prefollicle cells were labeled invarious lines. However, expression frequently varies fromstage to stage, not only in cell type but also in subcellularlocation, complicating the problem of accurate annota-tion. Currently, protein trap images within the ovary arebeing curated in the FlyTrap database. Because of therelative cellular simplicity of the germarium and de-veloping ovarian follicles, it may be possible to developtools for displaying expression patterns at single-cellresolution in this tissue. It will be particularly valuable toadd data from many other tissues and developmentalstages for these same lines, to facilitate comparisons.
Identification of insertions not predicted by genomeannotation: One of the surprising results of our studieswas the relatively high frequency of EGFP-positive linesthat were located at sites not predicted to fuse to anyannotated Drosophila transcript. However, similar re-sults were observed in previous studies of gene traptransposons. At least 44% of insertions analyzed byMorin et al. (2001) were not within annotated genes;moreover, the reading frame of insertions in genes was
Figure 4.—Studies of protein trap expressionWe compared the apparent intensity of GFP-protein staining in the GSCs with the RNA levelof the corresponding gene as determined by Af-fymetrix arrays (Kai et al. 2005). (A–F) The pat-tern of protein trap expression of the indicatedgene (see Table 4 for strain names). The expres-sion level from Affymetrix software (mean ofthree measurements) is given.
Protein Trap Lines in Drosophila 1529
not determined. In yeast, Ross-Macdonald et al. (1999)observed that while 1346 EGFP-positive insertions werein the correct frame, another 480 were not. Since mostlacked an alternative start site, they postulated that ahigher than expected frequency of translational frame-shifting may occur. In a recent study in the mouse, 24%of genes were trapped in more than one reading frame(De-Zolt et al. 2006). We also observed this phenome-non; however, our studies emphasized the difficulty ofdrawing final conclusions until the location of everyinsertion and the actual pattern of splicing within themutant strains have been characterized.
Sensitivity of gene traps: A potential limitation ofprotein trapping in vivo is that many gene products maybe expressed at levels so low that EGFP expressed at thesame level could not be detected above background.Only 20% of mouse secretory traps that are G418resistant express detectable CD2, even though theneophosphotransferase gene is fused to CD2 (De-Zolt
et al. 2006). Only 33% of b-geo lines resistant to G418express detectable lacZ. This probably indicates thatmany genes exist that generate enough neophospho-transferase to confer G418 resistance, but not enoughb-galactosidase to be scored as lacZ positive (De-Zolt
et al. 2006). Consistent with this, Ross-Macdonald et al.(1999) found that 415 of 1340 in-frame fusions (31%)could be detected above background by immunofluo-rescence. In contrast, Huh et al. (2003), who taggedcomplete proteins, detected signals above backgroundfor 4156 of 6029 (69%) genes. The system we employedis also designed to tag full-length proteins, and this mayhave enhanced its sensitivity.
The requirement that each line generate EGFPfluorescence in embryos might provide a limitation onthe number of genes that could be tagged. However, ourexperiments argue that this poses relatively little selec-tion on which genes can be fused. We found that genesexpressed in germline stem cells at a wide variety oflevels on the basis of microarray studies had been fusedin our collection of protein trap lines. There was a roughcorrelation between the levels observed using antibodystaining in these cells and the microarray results. Thiswould indicate that the protein trap methodology canpotentially be used to analyze thousands of diverseDrosophila genes.
Increasing proteome coverage: Our analysis revealedtwo major limitations of the current strategy for gener-ating protein traps using P elements. Despite theadvantages of embryo sorting, the inherent 59 bias ofP-element insertion (Bellen et al. 2004) greatly limitsthe rate at which new genes can be trapped. Many of theinsertions were recovered when an insertion occurred atan internal promoter that lies within a coding intron ofanother gene isoform. Many genes lack such alternativepromoters, so it will be necessary to use differentmethods to efficiently recover a more diverse collectionof protein trap strains.
At least two alternative approaches are worthy ofconsideration. First, it should be possible to take advan-tage of the extensive collection of P-element insertionsin Drosophila genes that have been generated by theBDGP gene disruption project and other members ofthe Drosophila community (reviewed in Matthews
et al. 2005). We calculate that �2000 genes already havean existing P-element insertion within a coding intron.Moreover, P elements can recombine into the sites ofexisting P elements in the presence of transposase (Sepp
and Auld 1999). Consequently, a protein trap allele ofeach of these genes could, in principle, be generated bycombining a protein trap insertion of the appropriatereading frame with the ‘‘target’’ gene insertion in asingle strain, ideally using inserts bearing scorable mark-ers and then screening for replacement.
Alternatively, transposons with a broader insertionalspecificity than the P element would be worthwhile.piggyBac elements are suitable for widespread mutagen-esis of Drosophila genes (Thibault et al. 2004) and asgene traps (Bonin and Mann 2004). Minimal sequencesfor piggyBac transposition were defined recently (Li et al.2005). We obtained several hundred piggyBac proteintrap insertions that express EGFP, but the piggyBac genetrap vector appeared to be less efficient, on the basis ofEGFP intensity and Western blotting, than P-elementgene traps in nearby locations (also see accompanyingarticle by Quinones-Coello et al. 2007, this issue). Newvectors containing different splice acceptor and donorsites should be tested within the context of a piggyBacelement. Several new transposons with diverse inser-tional specificities, including Minos (Arensburger et al.2005; Metaxakis et al. 2005), are also worthy of con-sideration. Continued efforts to provide greater cover-age within the Drosophila proteome are warrantedbecause of the exceptional utility of protein traps inanalyzing the development and physiology of multicel-lular organisms.
We thank Lynn Cooley for helpful discussions. We thank X. Morin,W. Chia, A. Hudson, R. Lehmann, and A. Handler for reagents. We aregrateful to the following people for their assistance with diverse aspectsof the project: Alison Pinder, Joseph Carlson, Martha Evans-Holm,Crista Sewald, Becca Sheng, Melanie Issigonis, Megan Kutzer, EmilySeay, and Dianne Williams. We thank the Howard HughesMedical Institute, Carnegie Institution, and the National Institutesof Health for support. M.B. was a fellow of the American Cancer Soci-ety. T.G.N. is a Howard Hughes Fellow of Life Sciences ResearchFoundation.
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Communicating editor: T. Schupbach
Protein Trap Lines in Drosophila 1531
