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. 2007 Mar;175(3):1505–1531. doi: 10.1534/genetics.106.065961

The Carnegie Protein Trap Library: A Versatile Tool for Drosophila Developmental 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 ‡, Allan C Spradling *,1
PMCID: PMC1840051  PMID: 17194782

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 enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600–900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

THE central challenge of postsequence genomics is to learn how an enhanced knowledge of genes, transcripts, and proteins can be applied to better understand the biology of multicellular organisms. Gaining an accurate picture of where and when metazoan genes are expressed remains a prerequisite for many such advances (Stathopoulos and Levine 2005). The discovery of distinctive, regulated programs of gene expression at a fine scale has the potential to reveal new cell types and substructures that make up tissues and the biological processes that govern their function. However, sensitive and widely applicable methods will be required to detect and distinguish developmentally programmed gene expression changes from those caused simply by cell cycling or environmental perturbation.

Several methods for analyzing gene expression within tissues are currently available. Particular cell types can sometimes be cultured in vitro into populations of useful size. However, isolated cells in artificial media frequently behave differently from cells in vivo interacting with precisely positioned neighbors in three-dimensional microenvironments. Another approach is to isolate tissue cells by flow sorting, microdissection, or laser capture and then determine their expression profiles in depth (reviewed in Espina et al. 2006). Visualizing patterns of gene expression within the intact tissues of transgenic organisms containing gene expression reporters may be the most general method (Tomancak et al. 2002). Epitope tagging, enhancer trapping, and gene trapping all have the added advantage that gene expression can subsequently be observed in living tissues, revealing dynamic processes that are largely beyond the reach of methods based on fixed material (reviewed in Herschman 2003; Dirks and Tanke 2006).

Protein trapping is a variation of gene trapping in which endogenous genes are engineered to produce under normal controls protein segments fused to a reporter such as GFP. The great potential of this technology has been extensively documented in yeast, where large collections of strains that each trap a different gene have been generated using transposable elements (Ross-Macdonald et al. 1999) or by homologous recombination (Huh et al. 2003). Extensive gene and protein trapping has also been carried out in cultured embryonic stem (ES) cells (Gossler et al. 1989; Friedrich and Soriano 1991), where fusions with more than half of annotated mouse genes have been recovered (see Skarnes et al. 2004). However, relatively few of these ES cell lines have so far been used to generate corresponding mouse strains where the versatility and sensitivity of the method for analyzing tissue structure can be tested.

Large-scale protein trap screens may also reveal new information about genome structure and function. Identifying in an unbiased manner locations throughout a genome where a coding exon can be expressed tests the accuracy and completeness of its annotation. Characterizing the splicing patterns that lead to normal or aberrant GFP expression tests the current catalog of transcript isoforms generated by alternative splicing. Moreover, by recovering insertions in the same gene that splice differently and produce GFP with varying efficiency, such a project might generate a data set useful for studying the determinants of splice site selection and transcript stability.

Drosophila provides a favorable system for applying gene traps to diverse developmental and genomic studies. The genome sequence has been extensively annotated on the basis of experimental data (Misra et al. 2002). Thousands of enhancer trap lines have been generated in large-scale transposon screens and culled of redundant strains by the gene disruption project (see Bellen et al. 2004). In contrast, producing Drosophila protein trap lines has remained difficult. Several hundred such lines were generated using a mobile GFP-containing exon flanked by both splice acceptor and donor sites (Morin et al. 2001; Clyne et al. 2003). However, the process was highly inefficient, with as few as 1 in 1500 progeny flies expressing GFP. Positive lines often contained more than one insertion, preferentially tagged a small number of hotspot loci, and tagged many sites not predicated to fuse the GFP exon in frame to any known coding region (Morin et al. 2001). Kelso et al. (2004) found that the recovery of lines could be increased by using an automated embryo sorter to select GFP-positive embryos and established a website, FlyTrap, to gather information on Drosophila protein trap lines. Consequently, we initiated a large-scale protein trap screen to increase gene coverage, test the genome annotation, and address some of the remaining technical difficulties in efficient line production.

Here we report the production of lines that trap 600–900 Drosophila genes, including 244 where one or more trapped proteins can currently be identified. Using the Drosophila ovary as a test system we confirm that protein trap lines reveal fine-scale details of developmentally regulated protein expression, making them exceptionally valuable discovery tools for a wide range of studies. Finally, mapping RNA splicing patterns downstream from >1200 insertions provides insight into how an added exon affects splicing and suggests how the production of protein trap lines can be expanded to cover a larger 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 utilized the pPGA, pPGB, and pPGC vectors described in Morin et al. (2001). These elements carry a mini-white transgene in the opposite orientation to an enhanced green gluorescent protein (EGFP) exon, which is composed of EGFP sequence, without start or stop codons, flanked by splice acceptor and donor sites from the Drosophila MHC locus. A, B, and C refer to the position of the splice sites within the first and last codons of the EGFP exon sequence. Previously used pPGA, pPGB, and pPGC third chromosome insertions (Morin et al. 2001) were remobilized in the presence of balancer chromosomes. New insertions that mapped to the CyO balancer chromosome, did not express EGFP, and exhibited remobilization rates off of the CyO balancer of at least 60% in single-pair mating assays were recovered and used as starting stocks in the screen (see below).

piggyBac protein trap vectors:

To make a shuttle vector for subcloning the EGFP exon into different transposable elements, the entire EGFP exons from the pPGA, pPGB, or pPGC plasmids were excised from the original P-element plasmids (kind gift of W. Chia) using EcoRI and PstI and subcloned into pBluescript (Stratagene, La Jolla, CA). These plasmids were then cut with EcoRV and KpnI, end filled using Klenow, and religated to themselves to create pBS-GFPA, pBS-GFPB, and pBS-GFPC. The resulting plasmids carry the EGFP exon sequence between a unique EcoRI site at the 5′ end and unique PstI, SmaI, BamHI, and XbaI sites at the 3′ end. New tagging sequences can be inserted between the splice acceptor and donor sites of the exon using unique NcoI and XhoI sites.

Two different piggyBac protein trap vectors (Figure 1) were constructed using pBac{D. m. w+} (Handler and Harrell 1999) (kind gift of A. Handler). The pBac{D. m. w+} plasmid was digested with ClaI to remove most of the mini-white sequence and a linker containing HpaI, XhoI, and SpeI sites was inserted in its place to form pBAC{ΔClaI}. To create pBAC{BglII-GFP}, the EGFP exons from pBS-GFPA, pBS-GFPB, and pBS-GFPC were subcloned into the BglII and MfeI sites of pBAC{ΔClaI}. To create pBAC{HpaI-GFP}, EGFP exon sequences were inserted between the MfeI and HpaI sites of pBAC{ΔClaI}. An intronless yellow transgene from the yellow-BSX plasmid (Bellen et al. 2004) was then subcloned into the unique SpeI site of both pBAC{BglII-GFP} and pBAC{HpaI-GFP} to form either pBAC{BglII-GFP; y+}, which has the EGFP exon and yellow transgene oriented away from each other, or pBAC{HpaI-GFP; y+}, which has the EGFP exon and yellow transgene pointing toward each other (Figure 1A). Both pBAC{BglII-GFP; y+} and pBAC{HpaI-GFP; y+} vectors carrying the EGFP exon in the A frame were transformed into y w flies using the phspBac helper plasmid (Handler and Harrell 1999) (kind gift from A. Handler).

Figure 1.—

Figure 1.—

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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. Rare GFP+ embryos (red circles) registering above a threshold value are diverted by the machine and later used to start individual cultures. (C) Scheme for characterization of putative protein trap lines (see text). (D) Classification of the general types of relationships between transposon inserts and the local genome annotation. Classes 1–4 consist of insertions in the appropriate orientation located within a codon intron (class 1), a noncoding transcribed region (class 2), an upstream genomic region (class 3), or an exon (class 4). For each class, the insert was either of the appropriate frame (subclass A) or of nonappropriate frame (subclass B) to fuse to the protein if splicing continued to the next annotated exon splice acceptor site. Class 5 consists of transposons inserted >0.5 kb from a correctly oriented annotated gene. (E) The structure of cryptic transcripts initiated within the Drosophila mini-white marker gene that contain an ATG codon and splice in frame to EGFP, thereby allowing expression independent of an endogenous transcript in some lines. (F) Western blot analysis of Dlg1 and eIF-4E protein production in control animals (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). Tissue culture cells expressing N-terminal or C-terminal fusions are found in the cytoplasm (center and right).

We created stable genomic sources of the piggyBac tranposase using P-element transformation vectors. To place the piggyBac transposase under control of the ubiquitin promoter, the piggyBac transposase ORF was excised from phspBac using BamHI and DraI and ligated into the BamHI and SmaI sites of the pCasper3-Up2-RX poly(A) P-element vector (Ward et al. 1998) (kind gift of R. Fehon), which carried a modified multiple 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 fragment containing both the Drosophila hsp70 promoter and piggyBac transposase ORF was ligated into the EcoRI and StuI sites of pCasper4 to form pP{hsp70-pBACtrans}. These vectors were used to transform y w flies, using standard P-element transformation techniques.

To test the activity of the piggyBac transposase transgenes, single-pair matings were set up using the pBAC{HpaI; y+}24.3 insertion, which mapped to the X chromosome, and pP{Ub-pBACtrans} or pP{hsp70-pBACtrans} stocks. The pBAC{HpaI; y+}24.3 insertion was mobilized in males that were then outcrossed to y w females. Phenotypically yellow+ males in the next generation were scored as new insertions. The pP{hsp70-pBACtrans} was able to remobilize the pBAC{HpaI; y+}24.3 insert in 43% (n = 30) of single-pair matings tested whereas the pP{Ub-pBACtrans} was able to remobilize the pBAC{HpaI; y+}24.3 insert in 48% (n = 21) of single-pair matings tested. The pBAC{HpaI; y+}24.3 insertion was remobilized in the presence of a CyO balancer chromosome. New insertions that did not express EGFP and mapped to the CyO chromosome were used in the pBAC-based protein trap screen.

Generation of embryos with novel transpositions:

We isolated new EGFP-expressing P-element insertions using the following genetic scheme:

graphic file with name GEN17531505sc1.jpg

New pBAC insertions were generated through a similar genetic scheme:

graphic file with name GEN17531505sc2.jpg

Hereafter, P-element and piggyBac element-based protein trap lines were treated the same. For the F1 cross several hundred males and females of the appropriate genotypes were mated in bottles to produce several thousand males in which the elements were mobilized for the F2 cross. These males were crossed to 8000–10,000 virgin y w females in a population cage. These virgin females were obtained using a virgining stock that carried a heat-shock-inducible hid transgene on the Y chromosome (kind gift of R. Lehmann). Two separate overnight embryo collections from each population cage were screened for EGFP expression. We limited the number of times we screened embryos from a particular cage to try to minimize the number of identical insertions recovered due to premeoitic insertion events.

Embryo sorting and line establishment:

We screened for EGFP expression in embryos using a COPAS Drosophila embryo sorter (Union Biometrica). Embryos were dechorionated in 50% bleach for 2.5 min and washed extensively with water. Dechorionated embryos were then washed into sorting solution (0.5× PBS, 2% Tween-20). With the exception of the sorting solution, the COPAS sorter was used according to the manufacturer's protocol, using the manufacturer's solutions. The sorter and sample pressures of the COPAS machine and embryo density were maintained so that the COPAS sorter screened 15–20 embryos/sec. The sorter used a 488, 514 nm multiline argon laser. EGFP fluorescence was detected using PMT1 set to 510 nm. Red fluorescence, used as a measure of embryo autofluorescence, was detected using a second PMT set to 580 nm. Baseline values for each fluorescent axis were set empirically using previously isolated fly strains that express low levels of EGFP and y w non-EGFP expressing embryos (Figure 1). Approximately 250,000 embryos were sorted in five 50,000-embryo batches per day. Sorted embryos were collected and washed in dH2O. All the embryos from a single batch were placed together in standard food vials. We estimate that ∼80% of the sorted embryos survived to adulthood. Sorted flies that survived to adulthood and did not carry the Ki, P{ry+t7.2; Δ2-3}99B or P{w+; pBACtrans} chromosomes were individually outcrossed to a y w stock. New lines that carried EGFP-expressing insertions that did not map to the starting CyO chromosome were maintained as stocks.

DNA sequencing, RT–thermal asymmetric interlaced PCR analyses, and prediction of fusion potential:

Genomic sequences flanking either P-element or piggyBac protein trap insertions were determined by members of the Lawrence Berkeley Lab group using an established protocol for sequencing inverse PCR products from genomic DNA (Bellen et al. 2004). Database software developed for the annotation of the Drosophila gene disruption project (Bellen et al. 2004) was used to manage the sequence data. Once the insertion site of a given protein trap line was determined, a FileMaker Pro database that contained information [version 3.2 of the Drosophila genome annotation (Misra et al. 2002)] for all Drosophila transcripts, exons and introns, and their reading frames was used to predict which gene(s) and transcript(s) were being trapped by a given protein trap insertion.

We developed a reverse transcriptase coupled thermal asymmetric interlaced PCR (RT–TAIL) protocol largely on the basis of methods used to determine T-DNA insertion sites in Arabidopsis (Singer and Burke 2003). This method allowed us to determine the mRNA sequence adjacent to the EGFP exon without using gene-specific primers. Total RNA was isolated from 15 adult flies using an RNAqueous-96 automated kit (catalog no. 1812; Ambion, Austin, TX). The samples were ground in 200 μl of sample buffer and spun for 5 min at 14,000 rpm. The supernatant was placed in a 96-well plate and 100 μl of 100% EtOH was mixed with each sample. The sample was transferred to the filter plate, washed, and then treated with Dnase I (Ambion) for 15 min. Rebinding buffer was added to each well of the filter plate, and the plate was washed extensively. The RNA was eluted off the filter and precipitated with 7.5 m LiCl solution (Ambion). The resulting RNA pellet was washed with 75% EtOH and then retreated with Dnase I for 30 min at 37°. Dnase inactivation reagent (Ambion) was added to the samples. The RNA samples were spun and the supernatant was transferred to a new plate. A detailed protocol is available upon request.

The following GFP-specific primers were used for RT–TAIL PCR:

  • GFP-For1, 5′-GGAGGACGGCAACATCCTGG-3′;

  • GFP-For2, 5′-CAACGTCTATATCATGGCCG-3′;

  • GFP-For3, 5′-AGACCCCAACGAGAAGCGCG-3′;

  • GFP-Rev1, 5′-GTCGTGCTGCTTCATGTGGTCG-3′;

  • GFP-Rev2, 5′-GACACGCTGAACTTGTGGCCG-3′;

  • GFP-Rev3, 5′-AGCTCCTCGCCCTTGCTCACC-3′ .

The arbitrary degenerate (AD) primers used in this study were originally described by Singer and Burke (2003) but are listed here for convenience:

  • AD3, 5′-AGWGNAGWANCAWAGG-3′;

  • AD4, 5′-STTGNTASTNCTNTGC-3′;

  • AD5, 5′-NTCGASTWTSGWGTT-3′;

  • AD6, 5′-WGTGNAGWANCANAGA-3′.

A pool of the AD primers was mixed according to Singer and Burke (2003).

The first round of RT–TAIL PCR was set up in 96-well format using a one-step RT–PCR kit (QIAGEN, Valencia, CA). For every reaction 5 μl of total RNA was mixed with 10 μl 5× buffer, 2 μl 10 mm dNTP solution, 1 μl GFP-For1 or -Rev1 primer, 12.5 μl AD primer mix, 2 μl enzyme mix, and 17.5 μl of dH2O. The reverse transcription reaction was carried out at 50° for 30 min. The sample was then heated to 95° for 15 min and then cycled for 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 carried out according to Singer and Burke (2003), using GFP-For2 or -Rev2 primers and GFP-For3 or -Rev3 primers, respectively, and regular TAQ DNA polyermase (Roche, Indianapolis). The PCR products of the tertiary reaction were treated with exoSAP (United States Biochemical, Cleveland) and sequenced using GFP-For3 or -Rev3 primers.

The RT–TAIL PCR protocol using the three GFP-For primers, which amplified off of the 3′ end of EGFP, consistently yielded better results than the same reaction using the Rev primers. Therefore most of the RT–TAIL PCR data define splicing products at the 3′ end of the EGFP sequence. To identify sequence fusing to the 5′ end of EGFP, we employed a 5′ RACE kit according to the manufacturer's protocol (Ambion), using the EGFP reverse primers listed above.

Analysis of protein expression in tissues:

Samples were dissected in Grace's medium, placed in 48-well plates outfitted with a nylon mesh bottom, and fixed in 4% paraformaldehyde buffered in 1× PBS for 10 min at room temperature. The plate was washed extensively with PBT (1× PBS, 0.5% Triton X-100, 0.3% BSA) and incubated overnight at 4° with rabbit anti-GFP antibody (Torrey Pines) (1:2000) in PBT. The samples were then washed extensively with PBT and incubated with goat anti-rabbit Alexa488 (Molecular Probes, Eugene, OR) (1:400) for 4 hr at room temperature. The samples were then washed with PBT, stained with 2 μg/ml of DAPI, and mounted in Vectashield (Vector Laboratories, Burlingame, CA). Images were collected using a Leica SP2 confocal microscope.

RESULTS

Generating a large initial collection of tagged strains expressing EGFP:

Our initial strategy was to generate a much larger number of lines containing new protein trap vector insertions than in previous screens and to institute additional technical improvements. Because of their proven utility, we used the same P-element-based protein trap vectors employed by Morin et al. (2001), but we also constructed a similar set of vectors with piggyBac (Figure 1A). As described in materials and methods, we set up crosses in small population cages to limit the recovery of clusters, utilized dominant markers to remove the transposase source from all new lines, and identified rare GFP-expressing embryos rapidly and sensitively using an automated embryo sorter (Figure 1B). This protocol allowed us to screen >60 million embryos over a period of 2.5 years, to identify >7500 “green” embryos, and to use each one to start an individual culture (see Table 1). Ultimately, 7404 strains were successfully established, maintained by selection for white+ eye color, and analyzed further as diagrammed in Figure 1C.

TABLE 1.

Project summary

Type No. %
Total setup 7404 100
CA 1344 18
CB 4000 54
CC 1870 25
piggyBac A 190 3
Aligned sequence 5720 77
Clusters 1550 21
Independent aligned 4170 56
Gene hits 2149 29
Spacing hits 2093 28
Different genes 1154 16
Protein traps 244
Enhancer traps 256
Novel gene/exon 50
Unclassified 300
Balanced stocks 878 12
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The same scheme was used with both transposons; however, in practice the piggyBac vectors were not nearly as efficient at generating EGFP-positive candidate lines as the P-element vectors (Table 1). P-element vectors typically exhibited 70% mobilization and generated ∼1 EGFP-expressing embryo per 1000 sorted. In comparison, the piggyBac vectors displayed nearly 50% mobilization, but they yielded only 1 EGFP-expressing embryo per 50,000 sorted. Thus, the piggyBac vectors were slightly less efficient at mobilization, but drastically less efficient at generating EGFP-positive lines upon insertion. Consequently, we soon abandoned attempts to generate large numbers of piggyBac protein trap insertions (Table 1), but continued to characterize the lines we did recover to learn if they would shed any light on the lower frequency of trapping observed.

Localizing insertions on the annotated genome:

To identify candidate proteins that may have been fused within individual lines, we determined the genomic DNA sequence flanking the insertion(s) in collaboration with the Berkeley Drosophila Genome Project (BDGP) gene disruption project (materials and methods). In most cases, the sequences from both the 5′ and 3′ vector end junctions mapped by BLAST analysis to a unique insertion site within the Drosophila genome sequence. Lines for which the sequencing reaction failed, the sequence matched repetitive DNA, or the 5′ and 3′ sequences differed (indicating that two or more insertions were present in the stock) were recycled back into the starting pool, and frequently a unique single insert was eventually identified. Altogether the insertions in 1375 C frame, 3172 B frame, and 1009 A frame P-element and 164 piggyBac A frame lines were localized to unique genomic sites.

Knowing the genomic location of an insertion allowed us to predict which transcripts would incorporate the EGFP exon and whether they would undergo splicing and translation into a functional fusion protein. First, we removed ∼1550 duplicate lines derived from premeiotic clusters that were identified because they bore insertions identical in position and orientation to those in one or more sibling lines. Of the 4170 independent lines remaining, 2149 (52%) were associated with a gene correctly oriented for possible fusion (i.e., located between −500 and the 3′ end). We also classified the ways an insert can be located relative to its closest annotated transcript into general categories as diagrammed in Figure 1D and classified all the lines (Table 2).

TABLE 2.

Line types

Lines
Type Code N %
Coding intron, in frame 1A 1337 23
Coding intron, out of frame 1B 173 3
Noncoding intron, in frame 2A 29 1
Noncoding intron, out of frame 2B 429 8
1–500 bp upstream, in frame 3A 56 1
1–500 bp upstream, out of frame 3B 756 13
Exon 4 804 14
>500 bp to next oriented gene 5 2093 37
Total 5677 100
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See class definitions in Figure 1D.

Expression of a fusion protein is expected when the GFP exon resides between two coding exons within an intron of matching reading frame (class 1A). Such insertions made up only 23% of the total localized insertions and defined 192 different genes (Table 4). Forty percent of insertions were close to an annotated gene but were not predicted to express the EGFP exon (classes 2–4), while 37% of the lines were not located within 0.5 kb of a correctly oriented gene. EGFP production from these lines might be explained by the use of unannotated genic elements, by noncanonical splicing events, or by the presence of a second insertion at a canonical site.

TABLE 4.

Identified trapped proteins

Gene Line Sitea Chr Strand Phenotype T1 Insert Met Stop T2 Insert Met Stop T3 Insert Met Stop T4 Insert Met Stop Type
14-3-3ɛ CA06506 14068962 3R + Lethal RA 1.5 1 4 RB 1.5 1 4 RC 1.5 1 4 RD 1.5 1 4 1A
26-29-p CA06735 13987313 3L + hv RA 0.5 1 3 3A
Acon CC00758 21169155 2L + Lethal RB 1.5 1 4 1A
Actn CC01961 1927332 X − hv RB 2.5 2 10 RA 2.5 2 10 RC 2.5 2 10 1A
AGO1 CA06914 9841633 2R − hv RC 3.5 3 8 RA 3.5 2 7 RB 0.5 2 7 1A
Alh CC01367 2935653 3R − hv RA 5.5 2 10 RD 5.5 2 10 1A
apt CC01392 19468319 2R + hv RB 1.5 1 5 RD 1.5 2 6 RE 1.5 3 6 RC 1.5 1 5 1A
apt CC01186 19473808 2R + hv RB 1.5 1 5 RD 2.5 2 6 RE 2.5 3 6 RC 1.5 1 5 1A
arg CB03579 417662 X + hv RA 3.5 1 5 1A
Argk CB05492 9051633 3L − Lethal RA 3.5 2 4 RB 2.5 2 3 1A
Argk CB03789 9056078 3L − Lethal RA 2.5 2 4 RB 0.5 2 3 1A
Atpα CC00319 16783418 3R + Lethal RC 1.5 3 10 RA 1.5 1 10 RB 0.5 2 9 RD 0.5 3 6 1A
baz CC01941 17072582 X + hv RA 1.5 1 7 1A
bel CC00869 4485350 3R − hv RA 1.5 1 4 1A
Best1 CB02354 5996196 3R − hv RA 1 2 7 4
βTub56D CC02069 15338563 2R − Lethal RB 1.5 1 2 RC 1.5 2 2 RD 1.5 2 2 1A
bon CB02667 16418866 3R + Semilethal RA 0.5 1 10 3B
Bsg CA06978 8104393 2L + hv RB 2.5 2 7 RA 2.5 2 7 RD 2.5 2 7 RC 2.5 2 7 1A
bun CB03431 12482943 2L − hv RA 3.5 1 5 RB 1 1 3 RD 1.5 1 3 RE 2.5 2 4 1A
Cam CC00814 8149270 2R + Lethal RA 2.5 2 5 1A
CAP CA06924 6190378 2R + hv RI 9.5 1 14 RH 7.5 3 12 RJ 8.5 2 13 RG 7.5 3 12 1A
CAP CA07185 Transposon 2R + hv RI/RF 3.5 1 14 1A
Cat CC00907 18815951 3L + Lethal RA 1.5 1 3 1A
Cg CC01469 10063184 2R + hv RD 1.5 1 10 RB 1.5 1 11 RC 2.5 3 11 RA 2.5 2 11 1A
CG10724 CA07499 13406231 3L + hv RA 1.5 1 7 RB 1.5 1 7 1A
CG11138 CA06844 12472570 X − hv RC 1.5 1 4 1A
CG11255 CB04917 13015302 3L − hv RA 1.5 1 3 1A
CG11266 CC01391 7033157 2L + hv RA 2.5 2 6 RD 2.5 8 9 RG 2.5 7 8 RF 1.5 6 7 1A
CG11963 CC06238 4764704 3R + Semilethal RA 2.5 2 9 1A
CG12163 CC00625 1076935 3R − hv RA 1.5 1 6 RB 1.5 1 5 1A
CG12785 CC06135 12098103 3R − Lethal RA 1 1 6 4
CG13920 CC01646 1650510 3L + Lethal RA 1.5 1 3 1A
CG14207 CB02069 19502492 X + Lethal RA 1.5 1 4 RB 1.5 1 5 1A
CG1440 CA07287 8331699 X + hv RA 1.5 1 5 1A
CG14648 CA06610 229231 3R + hv RA 1.5 1 6 RB 1.5 3 6 1A
CG14656 CA06996 624015 3R − Lethal RA 2.5 1 3 1A
CG15926 CB04063 12365107 X + hv RA 0.5 2 5 3A
CG1600 CB03410 3416244 2R − hv RA 1.5 2 3 RC 1.5 1 3 RB 1.5 2 3 1A
CG17273 CC01294 16654986 3R − Lethal RA 1.5 1 5 1A
CG17646 CB02833 1737431 2L + hv RB 1.5 2 12 RA 0.5 2 12 2B
CG1888 CB02075 5434043 2R − hv RA 1.5 1 2 1B
CG1910 CC01491 27573862 3R + hv RB 1.5 2 4 RA 1.5 1 4 RD 0.5 2 4 1A
CG3036 CA06801 4894157 2L + hv RA 2.5 2 7 1A
CG31012 CA06686 26640622 3R + hv RC 1.5 1 6 1A
CG31012 CA06810 26646037 3R + hv RC 4.5 1 6 RD 1.5 1 3 1A
CG31694 CA07748 2873276 2L − hv RA 1.5 1 5 1A
CG32062 CC00511 10515608 3L + hv RB 2.5 2 13 RD 2.5 2 14 1A
CG32423 CC00236 5177892 3L − hv RA 3.5 3 10 RD 2.5 2 9 RB 3.5 3 10 RC 1 2 8 1A
CG32479 CA06614 882041 3L + Lethal RA 4.5 2 10 1A
CG32486 CC00904 3060297 3L − hv RD 1.5 1 8 1A
CG32560 CA06772 17632469 X + hv RA 3.5 1 8 1A
CG3287 CB04483 2699967 2R + hv RB 2.5 2 5 RC 1.5 1 4 1A
CG33936 CC01586 5176796 3R + Lethal RA 2.5 3 6 RB 1.5 2 5 2B
CG3810 CA07694 1802294 X − hv RC 1.5 2 4 RB 1 2 4 RA 0.5 1 3 2B
CG3939 CA07562 3069871 X + hv RA 1.5 1 2 1A
CG5059 CA06926 20509742 3L − hv RA 1.5 1 5 RC 1.5 2 5 RB 1.5 1 5 RD 1.5 2 5 1A
CG5060 CC00526 16079041 3R + Lethal RA 1.5 1 7 1A
CG5130 CB03619 20486552 3L − hv RA 1.5 2 4 RB 1 2 4 2A
CG5174 CA07176 14309292 2R + hv RJ 1.5 1 6 RI 1.5 1 6 RA 1.5 1 6 RH 1.5 1 5 1A
CG5392 BA00207 15001227 3L − RA 2.5 2 8 1A
CG6151 CA07529 15808775 3L − Lethal RA 2.5 1 5 RC 2.5 1 5 RB 2.5 1 5 1A
CG6330 CB03223 22779020 3R − Lethal RB 2.5 2 6 RA 0.5 1 5 1A
CG6416 CC00858 8627040 3L + Lethal RE 3.5 1 9 RF 3.5 1 9 RA 2.5 2 8 RG 2.5 2 5 1A
CG6424 CC00677 13604099 2R − hv RA 2 3 4 RB 0.5 2 3 4
CG6783 CA06960 7392313 3R − hv RB 1.5 1 3 RC 1.5 1 3 RA 1.5 2 4 1A
CG6854 CA07332 15099355 3L + Lethal RC 1.5 1 4 RA 1.5 2 4 RB 1.5 2 5 1A
CG6930 CA06556 7597410 3R − hv RA 0.5 1 5 RB 2.5 2 6 RC 1.5 1 5 1A
CG6945 CC00864 15088263 3L − Lethal RA 0.5 3 3 3A
CG7185 CC00645 8308089 3L − hv RA 1.5 1 7 1A
CG7484 CB04101 17647286 3L + hv RB 0.5 1 3 3A
CG8209 CB02086 7969693 3L + hv RA 1.5 1 3 1A
CG8213 BA00169 4863309 2R − RA 1.5 1 8 1A
CG8351 CA07228 4631299 3R + Lethal RA 4.5 1 5 1A
CG8443 CA06604 12075198 2R + hv RA 1.5 1 7 1A
CG8552 CA07352 8159580 2L − hv RA 0.5 1 7 3A
CG8583 CA06603 7354388 3L + hv RA 1.5 1 4 1A
CG8920 CC00825 16208199 2R + hv RC 2.5 2 8 RB 2.5 2 8 RA 1.5 2 4 1A
CG9331 CB04962 20824559 2L + hv RB 1.5 2 5 RD 1.5 2 5 RC 1.5 2 6 RE 1.5 1 5 1A
CG9772 CB02188 163496 3R − hv RB 1.5 1 5 RA 0.5 1 5 RC 0.5 1 2 1A
CG9796 CC00817 9226998 3R − hv RA 1.5 1 4 1A
CG9894 CC00719 2755189 2L + hv RB 2.5 2 4 RA 1.5 1 3 1A
Cp1 CC01377 9852057 2R + hv RB 2.5 2 4 RA 2.5 2 4 RC 2.5 1 4 1A
Crc CA06507 5456158 3R − hv RA 2.5 1 4 1A
Crp CB03073 16280297 2L − hv RA 1.5 1 3 1A
CycB CC01846 18694008 2R − hv RA 1.5 1 5 RD 1.5 1 5 RB 1 1 5 RC 0.5 1 4 1A
Dek CC00921 12743940 2R + hv RA 1.5 1 11 RC 1.5 1 11 RB 0.5 1 10 RD 1.5 1 9 1A
Dek CA06616 12744777 2R + hv RA 2.5 1 11 RC 2.5 1 11 RB 1.5 1 10 RD 2.5 1 9 1A
desat1 CC01694 8269738 3R + hv RA 1.5 2 5 RC 1.5 2 5 RE 1.5 2 5 RB 1.5 2 5 2B
Df31 CB02104 21629393 2L − hv RB 2.5 2 4 RA 1.5 1 3 RF 1.5 1 3 1A
dlg1 CC01936 11286274 X + hv RF 5.5 1 9 RB 6.5 2 17 RH 5.5 2 16 RE 2.5 2 13 1A
DLP CA06573 6480860 2L + hv RA 0.5 1 4 3A
Doa CB03889 24717483 3R + hv RA 3.5 1 13 RB 3.5 2 13 RE 0.5 2 12 RF 3.5 1 13 1A
dom BA00164 17211471 2R + RD 1.5 2 15 RA 1.5 2 14 RE 1.5 2 11 2A
Dp CA06594 9111298 2R + hv RA 1.5 1 9 1A
drl CC00251 19190343 2L + hv RA 0.5 1 4 3B
Ef2b CC01924 21681694 2L − Lethal RA 1.5 1 5 RC 1.5 2 5 RB 1 2 5 1A
eff CC01915 10565092 3R − Lethal RA 2.5 2 6 1A
eIF-2β CC06208 12519527 3L − Lethal RA 1.5 1 3 1A
eIF3-S9 CB04769 13423919 2R + hv RB 1.5 2 5 RA 1 1 4 2A
eIF-4a CB03721 5982474 2L + Lethal RA 1.5 1 5 RC 1.5 1 5 RB 0.5 1 5 RD 0.5 1 5 1A
eIF-4E CC00392 9395078 3L − hv RD 1.5 2 6 RB 1.5 2 6 RC 1.5 1 5 RA 1.5 2 6 1A
eIF-5A BA00155 19945688 2R + RB 1.5 2 4 RA 1.5 2 4 2A
eIF-5C BA00280 1425192 3R − RA 1.5 2 8 RC 2.5 3 9 RF 1.5 2 8 RD 1.5 2 8 2A
Eip63E CA06742 3569399 3L + hv RD 2.5 1 11 RE 3.5 2 12 RA 4.5 3 12 RB 3.5 2 11 1A
Elf CA06515 12435789 2L + hv RA 1.5 1 7 1A
eRF1 CB03931 20342600 3L + Lethal RC 2.5 2 8 RB 2.5 2 8 RE 2.5 2 8 RG 2.5 2 8 1A
Fas2 CB03613 4029454 X − hv RA 9 2 10 4A
fax CC01359 16403817 3L − Lethal RA 1.5 1 5 RC 1.5 1 5 1A
Fer1HCH CA06503 26212791 3R − Lethal RA 1.5 1 3 RB 2.5 2 4 RC 2.5 2 4 RD 2.5 2 4 1A
Fer2LCH CA07607 26215006 3R + Lethal RA 3 3 4 RB 3 3 4 RC 1 1 2 4
Fim CC01493 17185787 X − hv RA 1.5 1 5 RC 2.5 2 6 RD 0.5 1 5 1A
Fkbp13 CA07340 17385064 2R − hv RA 0.5 2 5 RB 1.5 1 5 1A
Fpps CB04937 7194698 2R − hv RA 1.5 1 6 1A
Fs(2)Ket CA07301 20735659 2L + hv RA 2.5 2 6 1A
Gdi CA07108 9493967 2L − Lethal RA 1.5 1 3 1A
gish CB02804 12106314 3R + hv RD 2.5 2 12 RB 2.5 2 12 RE 2.5 2 12 RA 0.5 3 12 1A
GlcAT-S CA07168 9616795 2L + hv RA 1.5 1 5 RB 0.5 1 5 1A
Gli CB02989 15762784 2L − hv RA 0.5 2 7 RB 0.5 2 5 RC 0.5 3 8 RD 0.5 2 7 3A
G-oα47A CA06658 6331783 2R + Semilethal RB 2.5 2 8 RC 2.5 3 9 RD 2.5 2 8 RE 2.5 2 8 1A
gp210 CC00195 1647884 2R + hv RA 0.5 1 19 5
HDAC4 CA07134 13172850 X − hv RA 2.5 2 14 RC 1.5 1 12 1A
heph CC00664 27763272 3R − Lethal RB 4.5 4 14 RA 4.5 4 14 RK 3.5 3 13 RH 5.5 5 16 1A
His2Av CC00358 22693293 3R + Lethal RA 2.5 1 4 1A
homer CB02121 6722933 2L − hv RA 1.5 1 6 RB 0.5 1 6 RC 1.5 1 7 1A
how CA07414 17881934 3R + Lethal RA 2.5 2 8 RB 2.5 2 7 RC 2.5 2 8 1A
Hrb87F CC00189 9485980 3R − hv RA 1.5 1 3 RB 1.5 1 3 1A
Hrb98DE CC01563 24425969 3R + hv RA 1.5 1 5 RE 1.5 1 5 RB 0.5 1 5 RC 0.5 1 5 1A
Hrb98DE CA06921 24427038 3R + Lethal RA 2.5 1 5 RE 2.5 1 5 RB 2.5 1 5 RC 2.5 1 5 1A
Hsc70Cb CB02656 14031299 3L + Lethal RA 2.5 2 6 RB 2.5 2 6 RC 1.5 1 5 1A
Imp CB04573 10702676 X − hv RF 2.5 2 6 RH 3.5 3 7 RG 2.5 2 6 RD 2.5 2 6 1A
Indy CC00377 18833638 3L − hv RB 1.5 1 9 RC 1.5 2 9 RA 0.5 2 9 1A
inx7 CB04539 6892590 X − hv RB 4.5 3 5 1B
jumu CC00294 6182245 3R + hv RA 1.5 1 3 1A
Jupiter CB05190 7430651 3R − hv RD 1.5 1 4 RH 1.5 1 5 RA 1.5 1 5 RE 0.5 2 6 1A
kay CC01156 25608214 3R + hv RA 1.5 1 3 RB 0.5 1 3 1A
kek1 CB02190 12822834 2L − hv RA 0.5 1 2 3A
kis CC01466 220632 2L − hv RA 12.5 2 18 RB 1.5 1 7 1A
kis CC00801 221924 2L − hv RA 12.5 2 18 RB 1 1 7 1A
l(1)G0084 CC01368 19525602 X − hv RA 4.5 2 12 1A
l(1)G0168 CC00492 15393000 X + hv RA 2.5 1 7 RB 0.5 2 6 1A
l(1)G0320 CA06684 9447368 X − hv RA 1.5 1 2 1A
l(2)08717 CA06962 14688632 2R − hv RB 2.5 2 5 RA 1.5 1 4 1A
l(3)02640 CA07460 1336285 3L + Lethal RA 2.5 1 4 1A
l(3)82Fd CA07520 1123169 3R − Lethal RL 7.5 3 19 RB 7.5 3 19 RJ 7.5 3 19 RF 1 1 13 1A
Lam CB03749 5543070 2L + Lethal RA 1.5 2 4 2A
LamC CB04957 10462132 2R − Lethal RA 1.5 1 4 1A
larp CC06230 24152038 3R − hv RB 1.5 4 6 RC 1.5 2 6 RA 1.5 4 6 RD 1.5 1 6 2A
Lsd-2 CA07051 14969607 X − hv RA 1.5 1 4 1A
M6 CA06602 21502447 3L + hv RA 1.5 2 5 2B
Map205 CC00109 27891553 3R − hv RA 1.5 1 2 1A
Mapmodulin CC01398 13756894 2R − hv RB 3.5 3 7 RA 2.5 2 6 1A
mask CC00924 20060119 3R + hv RA 1.5 1 16 RB 1.5 1 16 1A
Mdh CB04968 22978192 3R + hv RA 1.5 1 7 1A
me31B CB05282 10240237 2L + hv RA 1.5 1 5 RB 1.5 2 5 1A
Men CC06325 8545125 3R − hv RB 2.5 1 4 RA 2.5 1 4 1A
Mi-2 CA06598 19901556 3L − Lethal RA 1.5 1 5 RA 1.5 5 5 RB 1.5 5 5 1A
Mob1 CB04396 11546502 3L − hv RC 1.5 1 4 RD 1.5 1 4 1A
mod(mdg4) CA07012 17200382 3R − hv RR 4.5 2 5 RA 4.5 2 5 RF 4.5 2 5 RD 4.5 2 5 1A
mub CC01995 21909824 3L + hv RA 7.5 2 9 RB 7.5 2 9 1A
NetB BA00253 14596460 X − hv RA 3.5 2 9 1A
NFAT CA07788 13534781 X + hv RA 1.5 1 10 1A
Nlp CC01224 25831370 3R + hv RA 2.5 1 3 1A
Nmdmc CB02647 4873684 3R − hv RA 1.5 2 3 RB 1.5 1 3 RA 1 1 2 1A
Nrx-IV CA06597 12141797 3L + hv RA 1.5 1 12 RB 1.5 1 14 1A
Oda CC01311 Transposon 3L − Lethal RA 2.5 1 11 1A
Oda CB03751 8060372 2R − hv RA 1.5 1 2 1A
Orc2 CB04400 9789604 3R + hv RA 0.5 1 5 RA 0.5 2 4 3A
osa CC00445 13529472 3R − Lethal RB 4.5 2 16 RA 4.5 2 16 1A
Pabp2 CC00380 4019736 2R + hv RA 2.5 2 5 RB 2.5 2 5 1A
Past1 CB02132 8523601 3R + hv RA 1.5 1 4 RB 1 1 3 1A
Pde8 CA07101 19554469 2R + hv RA 3.5 2 19 RE 4.5 3 20 RB 0.5 2 18 1A
Pdi CA06526 15134669 3L − Lethal RA 1.5 1 2 RB 1.5 1 2 RD 1.5 2 2 RC 0.5 1 2 1A
Pdp1 CB02246 7847944 3L − hv RF 1.5 1 5 RB 1.5 2 6 RG 1.5 2 7 1B
Picot CA07474 12548787 2R − Lethal RA 2.5 2 6 1A
Pkn CC01654 5157995 2R − hv RB 2.5 2 10 RC 2.5 2 11 RF 2.5 2 10 RD 2.5 2 10 1A
Pli CB03040 19712573 3R − hv RA 2.5 2 10 1A
Pmm45A CB02099 4996761 2R − hv RB 1 1 4 RA 1 1 3 4A
polo CC01326 20303643 3L + Lethal RA 1.5 1 5 1A
psq CC01645 Transposon 2R + Lethal RB 3.5? 2 10 1A
Ptp10D CC06344 11538719 X + hv RB 2.5 2 14 RC 1.5 1 10 1A
pUf68 CA06961 1501291 3L − hv RC 3.5 4 6 RD 3.5 6 8 RA 2.5 1 5 RB 2.5 4 6 1A
pum CC00479 4983771 3R − Lethal RA 8.5 2 13 RD 8.5 2 13 RC 8.5 2 13 RB 6.5 1 11 1A
Rab11 CA07717 16937950 3R + Lethal RA 1.5 1 4 RB 2.5 2 5 1A
Rab2 CA07465 2584592 2R + Lethal RA 1.5 1 4 1A
Rm62 CB02119 1833559 3R − Mfsterile RE 1.5 2 6 RC 2.5 3 7 RD 2.5 2 6 RB 1.5 2 6 1A
RpL10Ab CB02653 11815918 3L + Lethal RA 0.5 1 3 RC 0.5 2 3 RB 0.5 1 2 3A
RpL13A CC01920 1449810 3R + hv RB 0.5 1 3 3A
RpL30 CB03373 19009261 2L − Lethal RB 0.5 2 3 3A
Rtnl1 CA06523 5000772 2L − hv RB 3.5 2 7 RE 2.5 1 6 RD 0.5 2 6 RA 1.5 1 5 1A
Rtnl1 CA06547 4997815 2L − hv RB 3.5 2 7 RE 2.5 1 6 RD 2.5 2 6 RA 0.5 1 5 1A
S6k CC01583 5802962 3L − hv RA 1.5 1 10 1A
Sap-r CA07241 26714539 3R − hv RA 1.5 1 7 RB 1 2 6 1A
sar1 CA07674 18184952 3R + Lethal RA 4.5 2 6 1A
scrib CA07683 22393784 3R + hv RC 10 2 14 4
sd CA07575 15712098 X + hv RB 3.5 3 12 RA 1.5 2 10 1A
Sdc CC00871 17362946 2R − hv RC 2.5 2 6 RA 2.5 2 7 RB 2.5 2 6 1A
Sec61α CC00735 6479544 2L − Lethal RA 2.5 1 4 1A
Sema-1a CA07125 8592539 2L + hv RA 1.5 1 20 1A
Sema-2a CA06989 12411885 2R + hv RC 2.5 2 15 RB 2.5 2 15 RA 2.5 2 15 1A
sgg CA06683 2536739 X + hv RB 2.5 2 10 RA 2.5 2 10 RE 2.5 2 10 RF 2.5 2 10 1A
Sh3β CC01823 7361764 3L − Lethal RB 1.5 1 2 RA 2.5 2 3 1A
Sin CC01921 21024944 3L + Lethal RA 1 1 3 4
sls CA06744 2107593 3L − hv RC 0.5 1 1 RA 2.5 2 14 1A
sm CC00233 15504045 2R − hv RA 2.5 2 10 RC 2.5 2 9 1A
smi21F CA07211 1119094 2L − hv RB 2.5 2 5 RA 1 2 4 1A
sno CC01032 13104608 X − hv RB 1.5 1 12 RA 1.5 1 3 1A
snRNP69D CB02932 12727708 3L − Lethal RA 0.5 1 2 3A
sop CB02294 9897503 2L − Lethal RA 1 2 2 4A
SPoCk CA06644 22766451 3L + hv RA 0.5 2 4 RC 0.5 2 5 3A
Spt6 CA07692 6162464 X + hv RA 2.5 1 6 1A
sqd CB02655 9470746 3R − hv RB 1.5 1 7 RC 1.5 1 6 RA 1.5 1 5 1A
stwl CA07249 14402679 3L − Lethal RA 1.5 1 2 1A
Su(var)2-10 CC02013 5004591 2R + hv RI 1.5 1 8 RH 1.5 1 7 RD 1.5 1 9 RC 1 1 8 1A
Surf4 CC01684 11171291 3R − Lethal RA 1.5 1 4 RB 2.5 2 5 1A
sws CC01711 7861288 X − hv RA 1.5 1 11 1A
Sxl CB05562 6986034 X − Semilethal RB 2 2 3 RC 1.5 1 6 RN 1.5 1 6 RO 1.5 1 8 1A
TER94 CB04973 5877016 2R + hv RA 1.5 1 5 RB 1.5 2 4 1A
Tm1 CC01710 11116123 3R + Lethal RB 3.5 2 10 RJ 3.5 2 10 RD 3.5 2 10 RG 3.5 2 10 1A
Tm1 CC00578 11117364 3R + Lethal RB 3.5 2 10 RJ 3.5 2 10 RD 3.5 2 10 RG 3.5 2 10 1A
tmod CC00416 26389239 3R + hv RE 2.5 2 7 RD 1.5 1 6 RF 2.5 2 7 RC 2.5 2 7 1A
tmod CA07346 26400579 3R + Lethal RE 6.5 2 7 RD 5.5 1 6 RF 6.5 2 7 RC 6.5 2 7 1A
Top1 CC01414 15214506 X + hv RA 1.5 1 8 1A
tra2 CC01925 10491357 2R − hv RC 3 3 7 RB 2.5 2 5 RA 2.5 2 6 RE 1 1 4 4
tral CA06517 12508905 3L + Lethal RA 1.5 1 7 1A
Trxr-1 CA06750 8137659 X + hv RA 1.5 1 4 RB 1 1 4 1A
Tsp42Ee CC01420 2903327 2R + hv RA 2.5 2 5 1A
Tsp96F CC01830 21707093 3R − Lethal RA 1 3 RA 1 1 5 1B
tsr CC01393 19932991 2R − Lethal RA 1.5 1 4 1A
Tudor-SN CC00737 262842 3L − hv RA 1.5 1 4 1A
tun CC00482 11681495 2R − hv RG 2.5 2 16 RA 2.5 2 16 RC 2.5 2 16 RE 2.5 2 15 1A
twin CA06641 20044629 3R − hv RB 4.5 2 7 RE 5.5 2 8 RC 5.5 1 8 RD 5.5 3 4 1A
Uev1A CA07496 5358821 3L − Lethal RA 1.5 1 4 1A
VAChT CA06666 14538229 3R + Lethal RA 2 2 2 RA 1.5 1 8 1A
Vha13 CA07644 15469816 3R − Lethal RA 1.5 1 3 1A
Vha16 CA06708 2518996 2R − Lethal RA 2.5 2 4 RB 2.5 2 4 RC 1.5 1 3 RD 2.5 2 4 1A
Vha26 CC01380 1417892 3R + Lethal RB 2.5 2 5 RA 1.5 1 4 1A
Vha55 CA07634 8452738 3R − Lethal RB 2.5 2 4 RA 1.5 1 3 1A
vib CB05330 15045962 3R − Lethal RA 2.5 2 9 1A
vkg CC00791 5019005 2L − hv RA 2.5 2 9 1A
vsg CA07004 9707771 3L + hv RA 1.5 1 2 RD 1.5 1 2 RB 2.5 2 3 RC 2.5 2 3 1A
xl6 CB03248 6918786 2L − hv RA 1.5 1 2 RA 0.5 1 2 1A
yps CA06791 12117267 3L − Lethal RA 1.5 1 4 1A
zip CC01626 20896845 2R − Lethal RB 2.5 2 14 RA 2.5 1 14 1A
Zn72D CA07703 16102655 3L − hv RA 4.5 3 5 RB 4.5 3 6 1A
CB02318 4638102 3R + hv 5B
CC01309 8022556 3L + hv 5B
CB02658 12522067 2R − hv 5B
CC01670 12944837 3R + hv 5B
CC01932 21856862 3R − hv 5B
CA07436 5644358 2R − sl 5B
CB03064 8536402 2L + hv 5B
CC00523 22806145 3R − hv 5B
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a

Annotation release 5.0.

The Drosophila genome annotation is highly supported by experimental evidence (Misra et al. 2002); hence the frequency of these discrepancies was surprising, but a similar outcome has been reported in previous protein trap screens using both yeast (Ross-Macdonald et al. 1999) and Drosophila (Morin et al. 2001). Some protein-coding genes may have been missed within cDNA libraries (Hild et al. 2003), and a substantial number of genes may contain unannotated far-upstream promoters and alternative translation start sites. There might be a large class of RNA genes that have escaped detection but that can drive expression of EGFP using cryptic start sites. Alternatively, the high selective pressure used to isolate EGFP+ strains may have led to the recovery of rare events in which the normal gene or transcript structure has changed.

Analysis of fusion transcripts:

We sequenced portions of the fusion transcripts to address how EGFP expression arises in lines of various classes. A limited number of 5′ RNA sequences were obtained by 5′ RACE analysis or by TAIL–PCR (see materials and methods). As expected, several lines in class 1A were found to initiate in normal exons upstream from the insertion site. However, we discovered several CB lines that spliced into the EGFP exon from the 5′ P-element sequences of the vector. The P-element promoter is highly efficient at enhancer trapping, and the entire first exon of the transposase gene is present in the vector along with the start of intron 1, which is in the frame compatible with CB lines. These lines were associated with nuclear localized EGFP, possibly due to fusion of the first exon of P transposase with EGFP. Further evidence of enhancer trapping was observed in the analysis of line CA07138. The EGFP RNA was fused to sequences, including an in-frame ATG start codon, derived by transcription and splicing from the noncoding strand of the mini-white transgene carried in the P-element vector (Figure 1E). These observations suggest that EGFP expression in a significant number of the lines depends on transcripts initiated from within the transposon itself by enhancer trapping rather than on EGFP exon addition by splicing into exogenous transcripts.

Analysis of downstream transcript sequences:

To gain additional information, we analyzed the sequences downstream from the EGFP exon from many lines in the collection by carrying out RT–TAIL–PCR in a 96-well format (materials and methods). 3′ sequences up to 700 bp in length and defining the location of one to six downstream exons were obtained for >1200 lines (Table 3). The pattern of downstream splicing allowed productive fusions to be identified and indicated lines that splice out-of-frame and likely become subject to nonsense-mediated decay (NMD) (Vasudevan and Peltz 2003). Fusions within lines of classes 2–4 often were predicted to encode a “linker peptide,” which might or might not include a stop codon, derived from the translation of a small segment of upstream nucleotides. The RNA analysis also revealed the presence of a second insertion in 14% of type 1A lines, but between 24 and 50% of the other classes. The second insertions found within class 2–5 lines were often valid protein trap alleles (class 1A) and were frequently the true source of the lines' EGFP production. This information allowed us to identify additional candidate fusions (Table 4), to correct many initial line classifications, and to more accurately estimate the total number of trapped genes (Table 1: 600–900). By the time lines were selected and balanced, secondary insertions or damage did not contribute substantially to the phenotypes reported in Table 4. Tests estimated the frequency of background lethal mutations among balanced, saved lines at 7–21%, similar to the best transposon screens (Spradling et al. 1999).

TABLE 3.

RNA analysis

Type Successful Confirmed DNA Second insert % confirmed
CA 316 224 50 82
CB 328 166 75 69
CC 572 165 72 70
Piggy A 13
Totals 1229 555 197 74
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Novel splicing suggests new genes and exons:

We compared the splicing observed downstream from the inserted exons with that of the genome annotation (Misra et al. 2002) to identify new Drosophila gene and transcript isoform candidates. To identify new candidate genes, we focused on lines inserted >0.5 kb from an appropriately oriented known gene and for which RNA sequence data were also available. In 114 of these 205 lines, the RNA sequence coincided with the position and orientation of the insertion and therefore indicated the splicing pattern downstream of the single EGFP exon. Most of the lines spliced to one or more novel exons. At least 8 probably correspond to unannotated genes because they match previous gene predictions (Hild et al. 2003) or are supported by EST data (see Table 4). Most of the remaining exons do not predict proteins with homologs in other species and represent either aberrant splicing events or novel or untranslated exons.

Similar analysis of 297 lines with intron insertions allowed us to test for novel exons and transcript isoforms. We examined 443 splicing events and identified a total of 35 (7.9%) that did not correspond to current gene models (Misra et al. 2002). Since at least some of these differences probably resulted from aberrant splicing induced by the insertions, this represents a maximum estimate of the fraction of unannotated genomic exons and emphasizes the high accuracy and completeness of current Drosophila gene models, at least for abundant transcript forms. Often, the RNA data indicated which isoform among several predicted to fuse in frame is likely to predominate in ovarian tissue. For example, we could determine that line CA06613 in ovarian tissue predominantly fuses the Su(Tpl) gene rather than Mi-2, in whose transcription unit it also lies in frame.

The nature of the noncanonical splices observed was interesting. The most common events (21/35) were for insertions in large introns to splice to a novel exon(s) prior to joining the predicted downstream exon. Some simply appear to define alternative isoforms that skip exons or utilize different exon combinations not previously documented. Some of these events may have been induced by the abnormal position of the EGFP exon within the primary transcript. However, several lines appear to define alternative isoforms because they utilize different combinations of known exons in no previously documented transcript isoforms. Three lines utilized 5′ start sites for exons that differed by 6, 21, or 27 bp from the annotated exon. The CC01473 transcript reads through an annotated exon into the adjacent intron and probably defines a novel alternate transcript 3′ end. Although we consider it likely that many of these differences reflect endogenous Drosophila gene expression, all of the candidate novel genes and transcript isoforms require independent confirmation in strains lacking protein trap insertions. Such tests were beyond the scope of our project.

Protein trap insertions likely vary in the fraction of the endogenous protein that is tagged with EGFP for a variety of reasons. First, in many lines only some of the multiple-transcript isoforms contributing to protein production are tagged by the insertion and fused in frame. Second, the splicing efficiency of the EGFP exon might vary due to its surrounding genomic context. To investigate this issue, we analyzed the protein products of tagged genes by Western blotting. The tagged proteins were easily distinguished from their wild-type counterparts on the basis of size and by probing with protein-specific and anti-EGFP antibodies (Figure 1F). In line CC01936 all three isoforms are predicted to incorporate the EGFP exon in frame, and nearly all of the ovarian Dlg1 protein incorporated EGFP as indicated by its mobility. A similar result was reported previously in the case of line CB02119 (Buszczak and Spradling 2006), where the precursors of five of six annotated transcripts are predicted to contain the insertion, although only two fuse in frame. In contrast, only ∼50% of the ovarian wild-type eIF-4E protein is tagged with EGFP (Figure 1F) despite the fact that six of seven annotated eIF-4E transcripts initiate upstream from the insertion site. These examples suggest that the EGFP incorporation level varies between genes in large part due to the tagging of a subset of gene isoforms that themselves display differing expression levels.

In contrast, we found little evidence of short-range context effects. Less than twofold variation in protein expression as measured by Western blotting with anti-EGFP antibodies was observed between lines with insertions at sites within the same intron (N. Srivali and A. Spradling, unpublished data). However, these experiments did reveal that insertions of the piggyBac-based vector consistently produced less EGFP protein than lines with the corresponding P-element-based vector that were inserted in the same intron (N. Srivali and A. Spradling, unpublished data). This suggests that some aspect of the structure of the piggyBac vector used compromised splicing efficiency.

Identification of protein trap and enhancer trap core collections:

To help identify a core set of valid gene trap lines we examined the EGFP expression patterns of many nonredundant lines in both the adult ovary and larval salivary gland. There was a strong correlation between insert location and the nature of the staining patterns observed. More than 95% of lines in class 1A, the in-frame fusions, produced patterned EGFP expression above background in at least some ovarian cells or in the salivary gland. In contrast, a much smaller, but still significant, fraction of lines in classes 2–5 also expressed EGFP in a regulated manner. Combining information on insert location, genome annotation, RNA transcript sequence, and EGFP pattern, we identified a set of 244 lines predicted to produce fusions between EGFP and 431 protein isoforms of 233 distinct genes (Table 4). These new protein trap lines express EGFP in a wide variety of cellular compartments under diverse developmental controls (see Figures 2 and 3).

Figure 2.—

Figure 2.—

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Protein traps for the study of protein subcellular localization. Patterns of subcellular localization of EGFP expressed from the following lines that trap the indicated genes were observed: (A) cytoplasmic, CA06607 (CG17342); (B) nuclear, BA00164 (dom); (C) enhancer trap nuclear, CB04353 (Dad) in stem cells and early cystocytes; (D) endoplasmic reticulum, CA06523 (Rtnl1); (E) extracellular, CA06735 (cathepsin 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).

Figure 3.—

Figure 3.—

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Protein traps for the study of developmental regulation during oogenesis. The expression in the ovary of various protein trap lines is shown to illustrate how they can be used to associate genes with developmental processes. (A) Schematic of an ovariole tip. The terminal filament (TF), cap cells (CpC), germline stem cells (GSC), cystoblast (CB), and escort cells (ES) are illustrated. (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) fusome, 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 expression in early germ cells. (Q) Control with little change, CC01961 (Actn); (R) GSC/CB enriched, CC06238 (CG11963); (S) GSC and early cyst enriched, CC01915 (Eff); (T) GSC and forming cyst enriched, CC01442 (EIF-4E).

A second major class of lines in the collection showed the properties expected of EGFP enhancer traps (Table 5). These CB lines were susceptible to enhancer trapping from the P-element promoter, were located mostly upstream of the annotated start site, expressed EGFP in nuclei, and the RNA analysis, if available, did not indicate fusion in frame downstream. The expression patterns of such insertions in well-characterized genes supported this interpretation. For example, line CB02030 in ptc showed strong expression in the inner sheath cells of the germarium (Forbes et al. 1996), while line CB04353 in Dad strongly expressed in the germline stem cells and immediately downstream germ cells (Kai and Spradling 2003; Casanueva and Ferguson 2004).

TABLE 5.

Enhancer trap alleles

Gene Line Sitea Chr Strand Phenotype T1 Insert Met Stop T2 Insert Met Stop T3 Insert Met Stop T4 Insert Met Stop Type
1.28 CB04492 2389425 2R + hv RA 0.5 1 1 3
4EHP CB05417 19934636 3R − hv RA 1 1 4 4
abo/l(2)06225 CB03272 10975307 2L − hv RA 0.5 1 2 3
Adf1 CB02276 2550455 2R − Lethal RA 2.5 1 4 1
Adk3/CG4674 CB04307 6715035 3R − hv RB 0.5 2 2 RA 0.5 2 2 3
aop CB02762 2178398 2L − Lethal RB 1 2 4 4
ATPCL/CG8370 CB04427 11888767 2R − hv RA 0.5 2 9 3
bic CB03855 8757727 2R + Lethal RA 0.5 2 2 RB 0.5 1 1 3
bnl CB02854 15662816 3R − hv RA 0.5 2 5 RB 0.5 2 5 3
boca CB02070 3454283 2R − hv RA 0.5 1 2 3
bocksbeutel CB03586 5416706 3R − hv RA 1 1 2 4
Brd CB02665 14965467 3L − hv RA 1 1 4
btn CB03501 18414077 3R + hv RA 1 1 1 4
CBP CB03762 7230965 X + hv RA 0.5 1 4 3
Cct1 CB02171 1546105 3L + hv RA 1 2 4 4
cdi CB05129 14919950 3R + hv RA 1 2 8 2 2
CG10225/Tbp-1 CB02343 19578503 3R + hv RA 0.5 1 3 3
CG10272 CB03567 2233083 3R + Lethal RA 1.5 3 9 RB 1 2 8 RC 1.5 3 6 RD 1.5 3 9 2
CG10399 CB04753 6960416 2L + hv RA 0.5 1 2 3
CG10444 CB05265 16161990 2R − Lethal RA 1 1 2 4
CG10863 CB05229 3942458 3L + hv RB 1 1 6 3
CG10990 CB02351 13648912 X + hv RA 1 2 4 5
CG11042 CB03720 9046478 X + hv RA 0.5 1 1 3
CG1116 CB03511 1045469 3R − hv RA 1 1 5 RB 1 1 4 RC 1 1 5 4
CG11382 CB02249 1103926 X + hv RB 0.5 1 1 3
CG11526 CB02228 3325883 3L + hv RA 1.5 2 3 RB 1.5 1 3 2
CG11537 CB03533 3143415 3L − hv RC 1 1 8 4
CG11638/CG32814 CB02337 920725 X + hv RA 1 2 5 4
CG11779 CB05200 14983800 3R + hv RC 1.5 1 5 RA 1.5 1 4 RD 1.5 1 4 1
CG11940 CB05024 19742560 X − hv RA 1.5 1 3 RB 0.5 2 3 1
CG12360 CB02135 8856387 3R + hv RA 0.5 2 5 RB 0.5 2 5 3
CG12367 CB05312 8033260 2R + hv RA 1 1 3 4
CG1240 CB02937 2767042 3L + Lethal RA 1 2 2 4
CG12418 CB05329 5700359 3R + hv RA 0.5 1 1 3
CG12744/cbx CB05091 5762817 2R + hv RA 1 1 2 RB 0.5 2 3 3
CG12797 CB03502 10742504 2R + hv RA 0.5 1 1 3
CG13295 CB04422 6068307 3L + hv RA 1 1 3 4
CG13895 CB04175 708235 3L − hv RA 0.5 2 3 3
CG14215 CB02255 19546304 X + hv RA 0.5 1 7 3
CG14430 CB02236 6879851 X + hv RA 0.5 1 1 3
CG14478 CB02133 13345190 2R + hv RA 1 2 2 4
CG14542 CB02373 21878549 3R − Lethal RA 0.5 1 6 3
CG14709 CB04397 7394886 3R + hv RA 0.5 1 8 3
CG1621 CB02042 3380602 2R − hv RA 0.5 1 2 3
CG1667 CB05477 5731280 2R + Semilethal RA 2 1 5 4
CG16708 CB04388 1193533 3R − hv RA 1.5 2 4 2
CG16817 CB02934 5503678 3R + hv RA 1 1 4 4
CG16971 CB03898 224106 3L + hv RB 1 2 3 RC 1 2 3 RD 1 2 3 4
CG17002/vimar CB05094 2873307 2R + hv RB 1 1 5 4
CG17090 CB03116 544248 3L + hv RB 1.5 2 10 2
CG17090 CB02270 544285 3L − hv RA 1.5 1 2 5
CG17323 CB02962 18823565 2L + hv RA 0.5 2 5 3
CG17836 CB05263 14749549 3R + hv RB 3.5 3 5 RA 3.5 3 5 RC 2.5 3 4 RD 0.5 2 3 1
CG1910/l(3)s1921 CB03702 27573214 3R − Lethal RB 1.5 2 4 RA 0.5 1 4 2
CG2051 CB03625 1614858 3R + hv RB 1 1 3 RA 0.5 1 3 RC 0.5 1 3 4
CG2186 CB05020 10751743 X − hv RA 1 1 7 4
CG2446 CB03703 11608741 X − hv RA 0.5 3 4 RE 1 3 4 RB 1 3 4 RD 1 2 3 3
CG2698 CB03026 3827319 3R + hv RA 1 2 8 4
CG2865 CB03023 2187549 X − hv RA 0.5 1 2 3
CG2926 CB02232 1414090 3R + hv RA 0.5 1 1 3
CG2974 CB04047 9980614 X − hv RA 0.5 1 2 3
CG30055 CB02739 8477121 2R + hv RA 1 1 1 4
CG30497 CB02106 3667177 2R − hv RA 2 2 3 4
CG31241 CB02140 14081632 3R + Lethal RA 2 2 2 4
CG31475 CB04600 15006356 3R + hv RA 0.5 2 4 3
CG31522 CB02693 279018 3R − hv RB 1 2 10 RC 1 2 2 4
CG3164 CB02987 129446 2L + hv RA 1.5 1 8 RC 1.5 1 8 RB 0.5 1 7 RD 0.5 1 7 1
CG31650 CB05467 5043131 2L − hv RA 1.5 2 4 RB 1 2 4 RC 1 2 4 2
CG31688 CB03570 20429045 2L + hv RA 1 2 6 4
CG31689 CB03239 2735300 2L + hv RC 1.5 2 11 RD 1.5 2 11 RA 1.5 2 11 RB 1.5 2 11 2
CG31782 CB03345 16716141 2L − hv RA 2.5 1 6 RB 2.5 1 7 1
CG32043 CB03247 9455668 3L + hv RB 2 2 5 RA 2 2 5 4
CG3209 CB05445 19956511 2R + hv RA 1 1 7 RB 1 1 6 4
CG32345 CB03988 628140 3L − hv RA 0.5 1 1 5
CG32436 CB02614 21340125 3L − hv RA 1.5 1 5 1
CG32436 CB04148 21340268 3L + hv RA 1.5 1 5 1
CG32486 CB03414 3070840 3L + Lethal RD 0.5 1 8 3
CG3321 CB05150 10153408 3R + hv RA 2 2 2 RB 2 2 2 3
CG33214 CB04173 21500637 3L − hv RA 1 1 6 4
CG33232 CB03740 2466875 3L + hv RA 0.5 3 9 3
CG33558 CB05689 2859128 2R − hv RA 0.5 2 16 3
CG33967 CB04401 10549498 3R − Lethal RA 1 1 9 4
CG33982 CB04965 15527849 3L + hv RA 1 1 1 3
CG3409 CB04412 2602327 2R − hv RA 1.5 3 7 2
CG34110 CB04263 21010670 3R + Lethal RC 2.5 1 9 1
CG3428 CB02131 9480857 3L − hv RA 1 1 3 4
CG3654/Uch-L3 CB04163 9470143 3L − hv RA 0.5 1 4 3
CG4091 CB02284 19589843 2R − Lethal RA 1 2 3 RC 1 3 4 4
CG4300 CB04249 12270328 3L − hv RA 1 1 4 RB 1 1 4 4
CG4570 CB02124 6682617 3R + hv RA 1 1 1 4
CG4612 CB05331 20413641 2R − hv RA 0.5 2 3 3
CG5381 CB04616 10321949 2L + hv RA 0.5 2 7 3
CG5543 CB04854 19696764 2R + hv RA 0.5 1 1 3
CG5548 CB05667 14957872 X − hv RA 0.5 2 2 3
CG5677 CB02054 20055723 3R − Lethal RA 1 1 1 4
CG6014 CB04785 21390388 3L + hv RA 1.5 3 7 2
CG6218 CB03213 11168745 3R + hv RA 1 2 5 4
CG6311/Nedd4 CB04145 17522938 3L + hv RC 1 1 5 4
CG6439 CB03836 17844824 3R − Lethal RA 1 1 6 4
CG6499 CB05192 11075733 3R − Lethal RA 2 1 5 4
CG6540 CB03922 18542228 X + hv RA 1 1 2 4
CG6770 CB02632 12046132 2L − hv RA 0.5 1 1 3
CG7110 CB04925 13399559 2L + hv RB 2 2 7 4
CG7228 CB03115 7994181 2L − hv RA 1 2 5 RB 1 2 5 4
CG7331 CB05154 4175563 3R + hv RA 1 1 1 4
CG7637 CB03644 6698443 2R − hv RA 0.5 1 2 3
CG8036 CB04958 4495764 3R + Lethal RB 2.5 3 4 RC 1.5 2 3 1
CG8092 CB05336 11101113 2R − Lethal RA 1 1 6 RB 1 1 3 4
CG8128 CB02087 15591543 X + hv RA 1 2 4 4
CG8206 CB04527 15634596 X + hv RA 0.5 1 1 3
CG8444 CB03837 5086366 3R + Lethal RA 1 1 3 4
CG8583 CB04752 7353717 3L − hv RA 1 1 4 4
CG9062 CB02056 7171458 2R − hv RB 1 1 8 4
CG9171 CB02706 5800187 2L − hv RA 1 4 8 RB 1 3 7 4
CG9328 CB03031 20797707 2L − hv RB 0.5 2 3 3
CG9591 CB05694 9508902 3R − Lethal RA 2 2 9 4
CG9666 CB04503 19257579 3L − Lethal RA 1 3 4 4
CG9699 CB02196 16581750 X − hv RA 2.5 2 5 RF 2.5 2 5 RB 2.5 2 5 RD 2.5 2 5 1
CG9821 CB03335 4646100 3R − Lethal RB 1 2 2 RA 1 2 2 4
CG9921 CB02890 16226793 X − hv RA 0.5 2 3 3
CG9924 CB03517 9852398 3R − hv RB 2.5 3 9 2
Chd64 CB03690 4122396 3L − hv RB 1 1 3 4
chrb CB05429 11480949 3L + hv RC 1 1 4 4
Cp190 CB04933 11100985 3R + hv RA 0.5 2 5 2
crol CB03039 11809411 2L − hv RD 1 4 8 RA 0.5 4 8 RB 0.5 4 7 RC 0.5 4 8 4
Csp CB02217 22260750 3L + Lethal RA 1 1 5 RB 1 1 7 RC 1 1 6 4
CtBP CB04073 8837940 3R − Lethal RA 1.5 1 6 RB 1.5 2 8 RC 1.5 2 6 1
CycB3 CB02981 20696520 3R − Lethal RA 1 1 1 4
CycD CB02618 15803675 X + hv RC 1 1 5 RB 1 2 6 RA 0.5 2 6 4
Dad CB04353 12882290 3R + Semilethal RA 2.5 3 8 RB 1.5 2 7 RC 1.5 3 8 1
dally CB03495 8820577 3L + hv RA 0.5 1 9 2
dap CB05233 5599791 2R + Lethal RA 0.5 1 3 RB 0.5 1 3 2
Dap160 CB04282 21142183 2L − Lethal RA 1.5 2 11 RB 1.5 2 10 2
Dcp-1/pita CB03160 19439026 2R + Lethal RA 1 1 3 4
Dek CB02288 12743725 2R − hv RA 0.5 1 11 RD 0.5 1 9 RC 0.5 1 11 RB 0.5 1 10 3
desat1 CB02105 8269738 3R + hv RA 1.5 2 5 RC 1.5 2 5 RE 1.5 2 5 RB 1.5 2 5 2
Dl CB02040 15151950 3R − Lethal RA 0.5 1 8 RB 0.5 1 6 3
Dp1/imd CB03754 14299509 2R − hv RA 0.5 3 7 RB 0.5 3 7 RC 0.5 3 7 3
Dref/RpL13 CB02226 9967309 2L − hv RA 1 1 4 4
drk CB02974 9389656 2R − Lethal RE 1.5 2 6 RC 1.5 2 6 RB 1.5 2 6 RA 1.5 2 6 2
dup CB04090 11268107 2R − Lethal RA 1 1 3 4
eas CB02620 16172362 X + hv RB 1 2 5 RA 1 2 5 RE 1 2 6 RD 0.5 3 7 4
edl CB04040 14561061 2R − hv RA 0.5 2 2 3
eIF CB02125 1426796 3R + Lethal RA 1 2 8 RG 1 2 8 RC 0.5 3 9 RF 0.5 2 8 4
eIF3-S10 CB05493 259579 3R − Lethal RA 1 1 4 RB 0.5 3 5 4
eIF5B CB05358 3460609 3L − hv RB 0.5 1 9 3
Eip75B CB05160 18007641 3L − Lethal RB 1.5 1 6 1
emc CB02035 749295 3L + hv RA 0.5 1 2 3
endoA CB05084 14732350 3R − Semilethal RA 0.5 1 2 3
Eno CB02039 1727775 2L − Lethal RB 3 3 4 RE 3 3 4 RC 3 3 4 RD 3 3 4 4
esg CB02017 15333865 2L + Lethal RA 1 1 1 4
fas CB02992 9510510 2R + hv RA 0.5 3 13 3
fj CB04634 14120268 2R + hv RA 1 1 1 4
flfl/CG9591 CB05447 9510094 3R + Lethal RA 1.5 2 11 RB 1.5 2 11 2
fok/neb CB05794 20085050 2L − Lethal RA 1 1 2 4
for CB02956 3632190 2L − Lethal RA 4.5 3 10 RB 2.5 1 8 RH 3.5 2 9 RI 4.5 3 10 1
fs(1)K10/kz CB02790 2136127 X − hv RA 0.5 1 2 3
ftz-f1 CB05043 18758843 3L − Lethal RB 1.5 1 9 1
Fur1 CB03489 21298814 3R − hv RA 0.5 4 12 RB 0.5 4 12 RC 0.5 4 11 RD 0.5 3 10 3
fw CB05224 11898010 X − hv RA 0.5 2 15 3
fz2 CB02997 19163698 3L − Semilethal RB 1 3 3 4
glec CB02364 17681968 3R − Semilethal RA 1 1 2 4
Glycogenin CB05177 17087979 2R + Semilethal RB 0.5 1 5 RA 0.5 1 3 3
Grip163 CB05139 11988609 3L − Lethal RA 4 1 5 4
grp CB04894 16683920 2L + hv RB 1.5 2 7 RC 1.5 3 8 2
GstE1 CB04956 14285871 2R + Lethal RA 0.5 1 1 3
GstS1 CB05261 12984903 2R − hv RB 1 2 5 RC 1 2 5 RA 0.5 2 5 4
gukh CB04444 14809830 3R − hv RA 1 1 6 RB 1 1 5 RC 1 1 6 4
Gyc76C CB03117 19783669 3L − Lethal RC 2.5 5 19 RA 1.5 4 18 RB 1.5 3 18 2
hdc CB05774 26103641 3R + hv RC 0.5 1 6 3
HLHm7 CB02243 21862637 3R + hv RA 0.5 1 1 3
HmgD CB02648 17600985 2R + hv RA 1.5 2 3 RB 1.5 3 4 2
HP1b CB02849 8975314 X + hv RA 2 2 2 4
Hr39 CB05039 21250790 2L + hv RC 2.5 3 5 RB 2.5 3 8 RA 2.5 3 8 RD 2.5 3 8 2
Hsc70-4 CB05603 11068412 3R − hv RA 1 2 2 RC 0.5 2 2 RE 0.5 2 2 4
Hsr&ohgr CB03168 17122190 3R + hv RA 0.5 1 1 RB 0.5 1 1 RC 0.5 1 1 3
IP3K1 CB02227 9782577 2L + hv RA 1 1 4 4
Irp CB02050 6238617 3R + hv RA 1 1 10 4
kuz CB02000 13550247 2L + Lethal RA 1 2 12 RB 1 2 12 RC 1 2 12 4
l(2)gl CB02331 18536 2L − hv RB 2.5 2 9 RA 1 3 10 RC 2.5 2 9 RD 1 3 9 1
lama CB05457 5348459 3L − hv RC 1.5 3 5 RB 0.5 3 5 4
LanA CB04172 6211152 3L − hv RA 0.5 1 15 3
LBR CB03173 17607992 2R − hv RB 0.5 3 4 RA 0.5 2 3 RC 0.5 1 2 3
lea CB02898 1420531 2L − Lethal RA 0.5 1 14 3
Lk6 CB02120 7590184 3R − hv RA 0.5 1 6 3
lola CB02888 6429215 2R − hv RC 0.5 2 6 RG 0.5 2 6 RH 0.5 2 6 RJ 0.5 2 6 3
Map60 CB03167 5478371 2R + hv RA 1 1 2 4
mbc CB04603 19608306 3R − Lethal RA 1.5 1 14 1
Mbs CB02150 16045108 3L − Lethal RB 1 2 18 RA 1 2 18 RC 1 2 19 4
MESR3 CB02595 18617241 2L + hv RA 0.5 3 4 3
MESR4 CB04813 13435844 2R − Lethal RA 0.5 2 3 3
mirr CB02689 12686547 3L + Lethal RA 0.5 1 5 RB 0.5 1 5 3
Mnf CB03632 11113376 3L − hv RA 1.5 2 9 RE 1.5 2 10 RD 1.5 2 9 2
Mocs1 CB04106 11063638 3L + hv RA 0.5 1 4 RC 0.5 1 4 RB 0.5 4 4 3
mod CB02172 27878341 3R + hv RA 3 3 6 4
mRpS17 CB03663 20061952 2R − hv RA 1 1 2 4
Myo31DF CB04377 10506773 2L + hv RA 1 2 10 RB 1 1 9 4
neb/fok CB04551 20085119 2L + Lethal RA 1.5 1 2 1
nes CB05687 19253774 3L + hv RA 1 2 4 RB 1 2 4 RC 1 2 4 4
Neu3 CB02076 10523038 3R − Lethal RB 1 1 8 4
NK7.1 CB02349 10198828 3R − hv RA 0.5 2 4 3
nmo CB02015 7972207 3L + RA 1 3 9 RB 1 2 8 RE 1 2 8 RC 1 4 7 4
nmo CB04635 7972439 3L − Lethal RA 1.5 3 9 RB 1.5 2 8 RE 1.5 2 8 RC 1.5 4 7 3
Nrg CB04883 8411401 X + hv RC 0.5 2 8 RB 0.5 2 8 RA 0.5 2 8 3
orb2 CB04897 8946768 3L − hv RD 2.5 3 6 RB 1.5 2 5 RC 0.5 2 5 2
Orc2 CB03773 9790734 3R − Lethal RA 2 2 4 4
Pak3 CB04117 12279337 3R − hv RA 1 1 2 RB 1 1 2 4
Pi3K21B CB05132 297734 2L + hv RA 0.5 1 3 3
Pka-C1 CB03724 9699251 2L − Lethal RB 1 2 2 RA 1 2 2 RC 1 2 2 4
PNUTS-RB CB04203 870488 2L − Lethal RB 1 2 5 RC 1 3 4 RD 1 1 5 4
ppa CB03551 18576512 2R − hv RA 0.5 1 1 3
Psc CB05083 8868241 2R − hv RA 1 2 5 4
ptc CB02030 4537352 2R + Lethal RA 1 1 6 4
pxb CB05625 11491866 3R − hv RB 0.5 2 5 RA 0.5 2 5 3
PyK CB04247 18194169 3R + Lethal RB 1.5 2 4 2
Rab1 CB03450 17094755 3R − Lethal RA 1 1 5 RB 1 1 4 4
Rab23 CB03781 1509120 3R − hv RA 1.5 2 3 2
rdgB CB03856 13656772 X + hv RA 0.5 2 12 RC 0.5 3 13 RD 0.5 2 12 3
Rga CB02139 1438302 3R − Lethal RA 1 2 8 RB 1 2 8 4
rgr CB02242 4487949 2R + hv RA 1 1 5 4
rho CB05698 1463699 3L + hv RA 0.5 2 4 3
RhoGDI CB05226 19922267 3L + Lethal RA 1.5 2 5 2
Rpd3 CB04302 4626763 3L + Lethal RA 1 1 4 4
sas CB03307 2988505 3R + Semilethal RB 0.5 2 10 3
Sc2 CB04638 3903512 3L + hv RA 1 1 4 4
Sep2 CB05018 16414054 3R + hv RA 1 1 2 4
SF1 CB05052 13366171 3R + hv RA 1 1 6 4
SF2 CB03028 12167740 3R − hv RA 0.5 1 4 3
sgl CB05101 6957887 3L − hv RA 1 1 3 4
shg CB02169 16944287 2R − Lethal RA 1 1 2 4
Sir2/DnaJ-H CB02821 13165875 2L + hv RA 1 1 2 4
siz CB03288 21027758 3L − Lethal RA 1 1 6 4
skd CB02029 21018904 3L − Lethal RD 1.5 2 13 RC 1.5 2 13 2
Sod CB03598 11106792 3L − hv RA 1 1 2 4
Ssdp CB02353 14022949 3R − Lethal RA 1.5 2 2 RB 1.5 2 2 RC 1.5 2 2 2
stg CB03726 25081026 3R − hv RA 1 1 2 4
stumps CB05360 10417302 3R + Lethal RA 0.5 1 5 3
stwl CB05223 14402982 3L + hv RA 1 1 2 4
tankyrase CB03249 21486905 3R − hv RA 1 1 7 4
tara CB02767 12063444 3R + hv RA 1.5 1 2 1
tara CB03523 12075785 3R − Lethal RA 1.5 2 5 RB 1.5 2 5 1
Tbh CB04045 7889488 X + hv RB 0.5 2 8 3
Tctp CB03738 7036007 3R − hv RA 1 1 1 4
Tom CB02307 14962360 3L + hv RA 0.5 1 1 3
trbl CB02744 20394721 3L − Semilethal RA 0.5 1 2 3
trn CB05114 13107142 3L + hv RA 0.5 2 2 3
trx CB05156 10108478 3R − hv RA 1.5 3 9 RB 1.5 3 8 RC 1.5 2 7 RD 1.5 2 8 2
ttk CB02274 27550755 3R + hv RE 1.5 2 5 RF 1.5 2 5 RB 1 2 5 RC 1 2 5 2
Ugt37c1 CB02900 12733228 2R − Lethal RA 0.5 1 1 3
Ugt86Da CB03314 6982675 3R + hv RA 0.5 1 3 3
Vha100-2 CB03404 14224630 3R − hv RB 1.5 2 6 2
VhaPPA1-1 CB02209 10729723 3R − Lethal RA 1 2 2 4
wun2 CB02267 5301760 2R + hv RA 0.5 1 6 3
Xbp1 CB02061 17031115 2R + Lethal RB 1 1 3 RA 1 1 2 4
Xe7 CB05610 1495043 3R + Lethal RA 1 1 8 4
Z4/CG12974 CB02305 21279245 3L + Semilethal RA 1 1 2 4
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a

Annotation release 5.0.

The characterization of a significant number of lines in the collection remains incomplete (Table 1). Many of these contain insertions located >0.5 kb from an appropriately oriented annotated gene but where RNA sequence data were not obtained. Others are inserted within genes at locations not predicted to generate protein fusions or gene traps. Some of these lines express EGFP in the ovary, and we cannot rule out that others express transcripts in other tissues. On the basis of the processing of previous lines in these same classes this suggests that a significant number of new enhancer traps and a handful of new protein traps could be sorted out from a larger number of lines with secondary insertions in already trapped genes. Consequently, the number of different genes trapped in the collection is likely to increase beyond the 600 or so currently characterized.

Even when a protein is tagged in frame, the insertion of the EGFP sequence is expected to disrupt its normal structure and localization some fraction of the time. For example, line CC01311 traps CG15015, the Drosophila homolog of mammalian Cip4, a modular protein that interacts with Cdc42 and helps to regulate the actin cytoskeleton (Aspenstrom 1997). The CC01311 P element is inserted between the first two coding exons and thus disrupts the FES/Cip4 domain of CG15015 (Figure 1G). The protein trap fusion product localizes to the nucleolus while transgenes of CG15015 tagged at either the very N or C termini localize to the cytoplasm when expressed in S2 cells. We observed that 3 other protein trap fusion products of 107 analyzed accumulated in the nucleus when they were expected to reside in the cytoplasm.

Diverse behavior of tagged proteins:

The 244 identified protein trap lines of the core collection exhibit extremely diverse patterns of EGFP expression, suggesting that proteins occupying a wide range of cellular compartments can be tagged in vivo. We observed many lines with EGFP fluorescence in the cytoplasm (Figure 2A) or nucleus (Figure 2B) as expected. Localization to intracellular membranous structures was also commonly seen, as illustrated by a trap in the ER structural component Reticulon-1 (Figure 2D). Gene trapping of secretory proteins is thought to be inefficient due to retention of the fusion proteins in the ER where the activity of the fusion gene may be affected (Skarnes et al. 1995). The full-length protein traps we constructed could label secreted proteins, as indicated by the extracellular localization of EGFP in CA06735, a fusion with CG8947, a Drosophila cathepsin K homolog (Figure 2E), and the membrane location of a trap in Picot, a phosphate symporter (Figure 2F). Proteins that are apically localized in polarized epithelia such as ovarian follicle cells were easily visualized, as observed for Bazooka (Par3) (Figure 2G). Subcompartmentalized nuclear proteins were also readily apparent. For example, a trap of the Stonewall (Stwl) HMG-related protein involved in germ cell chromatin organization (Clark and McKearin 1996) labeled nurse cell nuclei and the oocyte nucleus (inset) differently (Figure 2H). Fs(2)Ket, a protein involved in nuclear import, was localized to the nuclear periphery (Figure 2I). In some cases, cell-specific cellular compartments were labeled, such as in CA07051, which traps Lsd-2 and exhibited EGFP localization to lipid droplets that arise in late-stage nurse cells (Figure 2J).

These studies provide a high-resolution view of known protein locations in living cells and also identify many proteins that were not previously known to reside within these compartments. In addition, the value of protein trapping as a discovery tool was illustrated by the fact that we observed new patterns of localization as well. For example, the HDAC4 protein, fused by the CA07134 trap, labeled a small body often found in only one nurse cell within an egg chamber (Figure 2K). A spindle-like structure in young nurse cells was labeled with a protein trap in the polo gene encoding a mitotic kinase (Figure 2L). Antibodies specific for the trapped protein can be used to isolate and further investigate the proteins present in these novel structures.

Analysis of developmental regulation—ovarian cells:

All the lines in the core collection were characterized on the basis of their patterns of expression in germ cells and follicle cells during oogenesis. These experiments identified lines expressing in the major classes of somatic cells, including terminal filament cells (Figure 3B), cap cells (Figure 3C), escort cells (Figure 3D), profollicle cells (Figure 3E), and border and posterior cells (Figure 3F). Other lines expressed in germ cells of various ages, including some that were highly enriched in the oocyte nucleus (germinal vesicle) (Figure 3G). As in the case of subcellular compartments, these studies documented patterns of developmental expression for many genes that were not previously known. These genes become attractive candidates for study of their function in the corresponding processes.

Strikingly, the collection also revealed the likely existence of new cell types and novel biological processes previously unrecognized despite many years of study of ovarian biology. Line CC01646 traps the CG12920 protein and is expressed in a small subset of ovarian sheath cells that likely represent a novel cell type (Figure 3H). In line CC00523 we observed accumulation of Msn-EGFP preferentially at the center of midstage growing follicles (Figure 3I). It was not previously known that this region was the site of unique protein accumulation. Msn encodes a protein involved in Jun kinase signaling, suggesting that a special intercellular junction may assemble in this region to structurally organize the nurse cells. We observed a similar expression program (not shown) for the line CB03040 that fuses the Pli gene, encoding a protein associated with the NF-κB signaling response.

Developmentally specific subcellular structures, including the fusome (Figure 3J, arrow) and both somatic and germline ring canals (Figure 3K), were also labeled by rare lines. A general and extremely useful application of the collection is to identify new proteins that are associated with such structures and analyze the effect of mutations. For example, the preferential accumulation of Sec61 in the fusome observed here has been validated in recent studies (Snapp et al. 2004). Proof of principle experiments of this type that focus on the fusome will be described elsewhere.

Another valuable capacity of protein trap lines is the ability to follow important developmental processes at high resolution and in living cells. During oogenesis, at least four major clusters of chorion genes undergo specific gene amplification in stage 10B follicles, a process that can be visualized as small “amplification dots” of BrdU incorporation (Calvi et al. 1998). The amplifying genes specifically contain substantial amounts of replication initiation proteins such as Orc2 at this time, whereas normally Orc2 is found throughout the cell nuclei (Royzman et al. 1999). A protein trap line in Orc2 allows the amplifying loci to be directly visualized (Figure 3L). Inspection shows that the dots are not present in preamplification stage follicle cells but strongly label amplifying gene loci at stage 10B (Figure 3L, arrow).

The Drosophila oocyte represents an important model system for studying RNA and protein localization. Several biochemical and genetic studies have identified proteins enriched at either the anterior or the posterior pole of the oocyte (Lasko 2003; Wilhelm and Smibert 2005). Protein traps in genes identified in these studies, including EIF-4E (Figure 3M) and Tral (Figure 3N), faithfully recapitulate the localization patterns of their endogenous counterparts to the posterior pole of the oocyte (Wilhelm et al. 2003, 2005). Several other proteins tagged in the collection display posterior localization patterns including a trap in CG32423 (Figure 3O), a largely uncharacterized RNA-binding protein. In addition, a trap in CG6151 appears to be enriched at the anterior of the oocyte (Figure 3P). While future work will clarify the role of these proteins in oocyte patterning, these examples show that protein trapping can complement other approaches and be used to identify new components of localized RNP complexes within the cells.

Protein traps provide unique opportunities to analyze gene regulation during development in populations of cells that are difficult to isolate and in those that are sensitive to loss of cellular context. We illustrate the potential of this approach using the regulation of germ cell development within and just downstream from the germline stem cells (GSCs). Many protein trap lines, including CC01961 in Actn, showed uniform expression in GSCs, CBs, and developing germline cysts (Figure 3Q). However, it was possible to find other examples where expression levels between GSCs and early germ cells differed from those in other germ cells within the germarium. One of the most striking examples was line CC06238 that traps the putative Drosophila succinate CoA ligase gene. Expression was stronger in stem cells (and sometimes early cystoblasts) than in later germ cells as illustrated in Figure 3R. Several other genes were downregulated shortly after GSC division, including effete (Figure 3S), encoding the UbcD1 ubiquitin-conjugating enzyme that has been shown to affect germline cyst formation (Lilly et al. 2000). Another line whose EGFP expression was downregulated slightly later, at the completion of cyst formation, trapped the Drosophila eIF-4E gene (Figure 3T). Downregulation of a related gene CG8023 was previously observed at a slightly earlier time, during cyst divisions (Kai et al. 2005).

Studies on the limitations of current protein trap methods:

We also tested the sensitivity of the approach used here to detect Drosophila genes by looking at the expression of the lines in the core collection in germline stem cells. First, although lines were selected on the basis of expression in embryos, we found ovarian expression above background in >90% of lines in the core collection. However, this does not address whether many other genes exist that were fused but expressed EGFP at levels too low to detect in either tissue. Analysis of germline stem cell RNA by hybridization to Affymettrix arrays detected transcripts from ∼6500 Drosophila genes over an ∼1000-fold dynamic range (Kai et al. 2005). Although translational regulation and differential protein stability, not to mention differences in staining sensitivity between different preparations, would be expected to introduce potential variation, we were curious whether protein trap lines could detect stem cell gene transcripts across the full range of expression levels.

We observed a strong correspondence between these two measures of stem cell gene expression (Figure 4, A–E). Lines with very strong EGFP expression tended to have RNA levels at least 10-fold higher than lines with above background but relatively weak expression. These lines in turn had signals higher than most lines scored as below the level of detection on arrays. The correlation was not perfect; for example, some lines showed more EGFP expression in stem cells than might be expected from the microarray study (Figure 4F). The existence of such lines was not unexpected, because some lines likely still carry second insertions, and the microarray used was based on release 1 gene models. Overall, we could detect EGFP above background in GSCs from nearly all lines whose levels of mRNA are called as “present” on Affymetrix arrays (Kai et al. 2005). This suggests that protein trap lines are not limited to a relatively small number of highly expressed genes, but can be used to follow a large fraction of gene activity.

Figure 4.—

Figure 4.—

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Studies of protein trap expression We compared the apparent intensity of GFP-protein staining in the GSCs with the RNA level of the corresponding gene as determined by Affymetrix arrays (Kai et al. 2005). (A–F) The pattern of protein trap expression of the indicated gene (see Table 4 for strain names). The expression level from Affymetrix software (mean of three measurements) is given.

DISCUSSION

The Carnegie protein trap collection—a versatile research tool:

These experiments significantly expand the number of protein trap lines available for studies of gene expression within a complex multicellular animal (also see accompanying article by Quiñones-Coello et al. 2007, this issue). Our initial characterization of these lines extends previous documentation that the behavior of the EGFP-tagged protein often corresponds to the behavior of the protein to which it is fused. Moreover, we demonstrate that collections such as ours are exceptionally useful as tools of gene discovery. Candidate genes can be selected on the basis of the developmental expression, subcellular localization, or dynamic behavior of particular protein isoforms. The same line can subsequently be used to purify the protein and its associated complexes and to create deletions for further genetic analysis. The Carnegie collection is available 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:

Previously, gene and protein trapping in yeast has been used to estimate the fraction of proteins that are localized to various cellular compartments (Ross-Macdonald et al. 1999; Kumar et al. 2002; Huh et al. 2003). More than half of all proteins showed a simple localization to the cytoplasm or nucleus. Other subcellular structures labeled by tagged proteins in yeast included the plasma membrane, the ER, mitochondria, the lysosome, and the perixosome. We obtained similar results. The distribution patterns of EGFP-tagged proteins within Drosophila ovarian cells generally matched those of yeast and most known structures within egg chambers have now been labeled with at least one protein trap line (this study; Morin et al. 2001; Clyne et al. 2003). Moreover, the large size of ovarian cells often allowed us to distinguish the fine structure of several subcellular compartments labeled by EGFP fusion proteins generated in this screen.

Developmental regulation of protein expression:

Despite the fact that only one tissue was examined closely, a large number of proteins in the core collection were expressed and many were developmentally regulated. A diverse array of cell types within the germarium including the terminal filament, cap cells, escort cells, germline stem cells, and prefollicle cells were labeled in various lines. However, expression frequently varies from stage to stage, not only in cell type but also in subcellular location, complicating the problem of accurate annotation. Currently, protein trap images within the ovary are being curated in the FlyTrap database. Because of the relative cellular simplicity of the germarium and developing ovarian follicles, it may be possible to develop tools for displaying expression patterns at single-cell resolution in this tissue. It will be particularly valuable to add data from many other tissues and developmental stages for these same lines, to facilitate comparisons.

Identification of insertions not predicted by genome annotation:

One of the surprising results of our studies was the relatively high frequency of EGFP-positive lines that were located at sites not predicted to fuse to any annotated Drosophila transcript. However, similar results were observed in previous studies of gene trap transposons. At least 44% of insertions analyzed by Morin et al. (2001) were not within annotated genes; moreover, the reading frame of insertions in genes was not determined. In yeast, Ross-Macdonald et al. (1999) observed that while 1346 EGFP-positive insertions were in the correct frame, another 480 were not. Since most lacked an alternative start site, they postulated that a higher than expected frequency of translational frameshifting 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 phenomenon; however, our studies emphasized the difficulty of drawing final conclusions until the location of every insertion and the actual pattern of splicing within the mutant strains have been characterized.

Sensitivity of gene traps:

A potential limitation of protein trapping in vivo is that many gene products may be expressed at levels so low that EGFP expressed at the same level could not be detected above background. Only 20% of mouse secretory traps that are G418 resistant express detectable CD2, even though the neophosphotransferase gene is fused to CD2 (De-Zolt et al. 2006). Only 33% of β-geo lines resistant to G418 express detectable lacZ. This probably indicates that many genes exist that generate enough neophosphotransferase to confer G418 resistance, but not enough β-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 immunofluorescence. In contrast, Huh et al. (2003), who tagged complete proteins, detected signals above background for 4156 of 6029 (69%) genes. The system we employed is also designed to tag full-length proteins, and this may have enhanced its sensitivity.

The requirement that each line generate EGFP fluorescence in embryos might provide a limitation on the number of genes that could be tagged. However, our experiments argue that this poses relatively little selection on which genes can be fused. We found that genes expressed in germline stem cells at a wide variety of levels on the basis of microarray studies had been fused in our collection of protein trap lines. There was a rough correlation between the levels observed using antibody staining in these cells and the microarray results. This would indicate that the protein trap methodology can potentially be used to analyze thousands of diverse Drosophila genes.

Increasing proteome coverage:

Our analysis revealed two major limitations of the current strategy for generating protein traps using P elements. Despite the advantages of embryo sorting, the inherent 5′ bias of P-element insertion (Bellen et al. 2004) greatly limits the rate at which new genes can be trapped. Many of the insertions were recovered when an insertion occurred at an internal promoter that lies within a coding intron of another gene isoform. Many genes lack such alternative promoters, so it will be necessary to use different methods to efficiently recover a more diverse collection of protein trap strains.

At least two alternative approaches are worthy of consideration. First, it should be possible to take advantage of the extensive collection of P-element insertions in Drosophila genes that have been generated by the BDGP gene disruption project and other members of the Drosophila community (reviewed in Matthews et al. 2005). We calculate that ∼2000 genes already have an existing P-element insertion within a coding intron. Moreover, P elements can recombine into the sites of existing P elements in the presence of transposase (Sepp and Auld 1999). Consequently, a protein trap allele of each of these genes could, in principle, be generated by combining a protein trap insertion of the appropriate reading frame with the “target” gene insertion in a single strain, ideally using inserts bearing scorable markers and then screening for replacement.

Alternatively, transposons with a broader insertional specificity than the P element would be worthwhile. piggyBac elements are suitable for widespread mutagenesis of Drosophila genes (Thibault et al. 2004) and as gene traps (Bonin and Mann 2004). Minimal sequences for piggyBac transposition were defined recently (Li et al. 2005). We obtained several hundred piggyBac protein trap insertions that express EGFP, but the piggyBac gene trap vector appeared to be less efficient, on the basis of EGFP intensity and Western blotting, than P-element gene traps in nearby locations (also see accompanying article by Quiñones-Coello et al. 2007, this issue). New vectors containing different splice acceptor and donor sites should be tested within the context of a piggyBac element. Several new transposons with diverse insertional specificities, including Minos (Arensburger et al. 2005; Metaxakis et al. 2005), are also worthy of consideration. Continued efforts to provide greater coverage within the Drosophila proteome are warranted because of the exceptional utility of protein traps in analyzing the development and physiology of multicellular organisms.

Acknowledgments

We thank Lynn Cooley for helpful discussions. We thank X. Morin, W. Chia, A. Hudson, R. Lehmann, and A. Handler for reagents. We are grateful to the following people for their assistance with diverse aspects of the project: Alison Pinder, Joseph Carlson, Martha Evans-Holm, Crista Sewald, Becca Sheng, Melanie Issigonis, Megan Kutzer, Emily Seay, and Dianne Williams. We thank the Howard Hughes Medical Institute, Carnegie Institution, and the National Institutes of Health for support. M.B. was a fellow of the American Cancer Society. T.G.N. is a Howard Hughes Fellow of Life Sciences Research Foundation.

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