BACKGROUND
genetic trapping experiments have a long-standing history in drosophila functional genomics.
the classic "enhancer trap" screens utilised p-element-mediated insertion of the e. coli lacz gene to facilitate relatively unbiased discovery of tissue-specifically expressed genes and enhancers at a genome-wide level  <cit> .
in recent years, additional transposable elements such as piggybac  <cit>  or minos  <cit>  have found their way into research applications.
together, this set of transposons provides excellent tools for genome-wide genetic screens.
while p-elements and piggybac show some positional preferences towards insertion at the 5' regions of target genes, and thus have biased coverage with respect to the genome  <cit> , minos is reported to show a much more random genomic distribution of insertions  <cit>  and may be more suited to genome-wide screens.
the introduction of the green fluorescent protein  in research applications opened new avenues for enhancer trapping, since the expression of a fluorescent reporter can be directly observed and detection does not require an enzymatic reaction  <cit> .
a more recent advance in the gene trapping approach is the "protein trap" screen, which aims to create gfp-tagged versions of endogenous proteins under the control of the genes' native regulatory sequences  <cit> .
as is evident from the comprehensive recombination-based efforts in yeast, tagging endogenous proteins in vivo can have tremendous utility for genomics, cell biology and systems biology studies  <cit> .
protein tagging is likely to be of even greater untility in metazoans where many different cell types are present.
protein trap screens utilise artificial reporter-encoding exons to generate fluorescent fusion proteins by random integration into the genome.
the reporter is usually a gfp variant, flanked by splice acceptor/donor sites, and carried within a transposable element vector.
integration of the transposon within an intron in the correct orientation results in the transcription and subsequent splicing of the trapping exon into the mature mrna of the targeted gene.
if the trapping exon is in-frame with coding sequence of the host protein, a functional gfp-tagged version of the protein may be produced.
a comprehensive screen obviously requires vectors carrying targeting exons in all three reading frames, but even when multiple vectors are employed the isolation of bona fide protein traps is a relatively rare event.
following the pioneering work of morin et.
al.
<cit> , the results from a variety of drosophila protein trap screens have been published  <cit> .
in total, these studies have screened close to  <dig> million individual embryos or larvae, with only a small fraction of these generating lines that express the gfp tag.
the flytrap database  <cit>  reports  <dig>  fluorescent lines generated from the three largest protein trap screens performed to date  <cit> .
mapping of their insertion coordinates to release  <dig>  gene annotation shows that these represent  <dig> unique genes tagged with protein traps located within introns separating coding exons.
with the fly genome containing approximately  <dig>  protein-coding genes, the screens have hit less than 2% of known fly genes.
this is far from genome-wide coverage, clearly a desirable goal for comprehensive functional genomics studies.
the restricted success of protein trap screens is especially surprising given that approximately  <dig>  drosophila genes contain introns.
thus, in principle, approximately 80% of fly genes are accessible to protein trapping.
interestingly, although the overall number of unique lines generated in the different screens is relatively small, there is considerable overlap in the tagged genes recovered by the individual screens.
the low efficiency and high degree of overlap between the published screens suggests that there are limitations to the protein trap strategies currently in use.
here we attempt to identify and quantify these limitations, and suggest future strategies that may increase the repertoire of trapped genes.
we considered a number of potential factors that could bias protein trap screens, including transposable element integration hotspots, gene architecture, gene expression and protein structure.
we constructed a probability model that we used to predict a set of target genes with a high likelihood of successfully receiving a protein trap insertion.
our model predicts that approximately  <dig> of the genes encoded in the fly genome are permissive for p-element based protein trapping and of these,  <dig> genes have already been tagged in previous studies .
a similar analysis based on data from a more limited set of protein traps generated with piggybac vectors estimates approximately  <dig>  genes are permissive targets with this transposon, and about  <dig>  of these have not yet been tagged in previous studies.
comparing the predictions for both transposons we find that most potential as yet untagged p-element targets are also good piggybac targets .
due to the apparent importance of transposon insertion bias, it is likely that a transposable element such as minos, which exhibits a more random insertion preference  <cit> , may be a better vector for future random protein trap screens.
ultimately, it is likely that recombination or recombineering -based gene targeting techniques will need to be employed to achieve comprehensive coverage of the fly genome.
RESULTS
the minority of gfp trap lines are real protein traps
we retrieved insertion data for  <dig>  previously described protein trap lines from the flytrap database  <cit> .
the genomic insertion coordinates were available for  <dig>  of the lines and these were taken forward for further analysis.
the insertion sites from flytrap were translated to drosophila genome release  <dig> coordinates and mapped with respect to the release  <dig>  gene annotations taken from flybase.
the exon boundary and coding sequence information was obtained for each transcript of the relevant genes and, where available, the relevant frame information from the original protein trap screen was utilised.
together, these data established a catalogue of protein trap target genes based on the most recent genome sequence and annotation releases.
the compiled data are available  with a summary provided in table  <dig>
the updated insertion coordinates were mapped to genes from flybase release  <dig> , and the insertion annotations were updated accordingly.
the mapping of a small number of lines was ambiguous due to the insertion being in various features of different transcripts and/or different nested genes – in the cases where an intron between coding sequence was hit, the line was classified as 'cds intron', regardless of the other features hit concurrently.
some genes are represented by multiple lines, so the  <dig> fly lines hitting cds introns in fact amount to  <dig> unique genes hit.
a canonical protein trap is defined as an insertion residing in an intron within the coding sequence of a gene that, after splicing, allows the translation of the gfp reporter in frame with the endogenous polypeptide.
the recovery of enhancer traps in protein trap screens has been previously observed and determined to be due to the presence of an upstream start codon.
the start codon is within the transposase gene of the protein trap vector itself  <cit> , but has also been reported to result from a splicing event from the non-coding strand of the mini-white marker present in the vector  <cit> .
the updated release  <dig> coordinates and gene annotations confirm that bona fide protein traps constitute the minority of the  <dig>  reported lines .
of the  <dig>  fly lines for which coordinates were available,  <dig> are insertions entirely outside gene regions and of the  <dig> remaining lines that do insert within a gene model, only  <dig> are found in introns between coding exons.
these  <dig> insertions tag a total of  <dig> unique genes, reducing to  <dig> genes if frame information is taken into account.
the  <dig>  insertions in places other than the introns within coding sequence are considered to be enhancer trap type insertions rather than protein traps.
<dig> of the flytrap lines have insertions targeting gene regions outside their coding sequence.
interestingly, only  <dig> of such insertions are located in the 3' utr of genes with the remainder 5' to the gene start codon.
while this suggests that it is indeed likely that the p-element promoter and translational start sites facilitate enhancer trapping, some of the traps may be located in introns associated with unannotated 5' exons and thus may represent bona fide protein traps.
insertion biases of protein trap insertions
approximately  <dig>  protein-coding genes in the fly genome have at least one intron and therefore 80% of the drosophila gene repertoire is in principle amenable to gfp-tagging.
in practise, the fraction of recovered protein traps is clearly much lower, indicating that there are preferred target genes.
this becomes evident when the total of  <dig> gfp-tagged proteins found in the three published large-scale screens are compared .
while the largest screen  <cit>  successfully tagged a number of new proteins , most of the proteins tagged in the smaller screens are recovered in multiple studies.
68%  of the protein traps reported by morin et al.
<cit>  and 66%  of those reported by quiñones-coello et al.
<cit>  were also recovered in one or both of the other screens.
transposable element hotspots introduce the greatest bias in protein trap screens
the p-element constructs of morin et al.
<cit>  were used in the two other screens reported in flytrap and lines derived with these vectors represent the majority of the insertions reported in the database .
the remaining insertions were generated with a piggybac vector .
to explore the possibility that p-element insertion preference biases the recovery of protein traps, we compared the repertoire of genes recovered in the protein trap screens with all of the p-element insertions reported in flybase.
twelve genes were independently recovered using p-elements in all three protein trap screens and we found an average of  <dig> other p-element insertions in each of these genes .
these  <dig> genes alone are covered by  <dig> independent lines reported in flytrap, accounting for  <dig> % of all the lines in the database and over 13% of coding sequence insertions.
this observation suggests that there may be significant bias due to p-element insertion hotspots.
noteworthy, amongst the  <dig> targets shared between all protein trap studies is cg <dig>  which accounts for 10% of all "kg"  lines recovered in the gene disruption project  <cit> .
for all remaining protein trap targets generated using p-element vectors, the insertion location is strongly biased.
of the  <dig> genes containing p-element gfp trap insertions into coding sequence introns ,  <dig>  have other p-element insertions and only  <dig> genes have no previously reported transposon insertions.
the frequency of p-element insertions in genes isolated in the protein trap screens is significantly higher than observed within the approximately  <dig>  protein-coding genes that have reported p-element integrations .
in addition, within the protein trap tagged genes, the integration hotspots are localised within the tagged intron since these have, on average,  <dig>  other p-element insertions.
other introns within tagged genes have, on average, only  <dig>  p-element insertions .
two of the published protein trap screens  <cit>  also used piggybac-based vectors, because of their reported preference for at-rich integration sites such as introns  <cit> .
these pilot screens generated  <dig> lines of which  <dig> insertions result in protein traps in  <dig> unique genes.
interestingly, the same bias towards previously isolated transposon targets is also found in the piggybac set  with  <dig> out of  <dig> genes containing previously reported transposon insertions:  <dig> of the  <dig> have other piggybac insertions.
a total of  <dig> previously reported piggybac insertions are associated with the protein trapped genes recovered in the piggybac screens : a comparable level to that observed in p-element screens.
the high frequency of other transposable element insertions associated with genes recovered in the protein trap screens indicates that these genes, and more specifically their tagged introns, are genomic hotspots for transposon insertion.
gfp traps selectively target longer introns
due to the much larger number of p-element protein trap lines reported in flytrap, we concentrate the majority of our further analysis on the differences between genes generally susceptible to p-element integration and genes recovered in the protein trap screens, to identify factors that may influence recovery of protein traps.
the average length of the  <dig> genes successfully tagged by p-element protein traps is  <dig>  kb, significantly higher than the  <dig>  kb average size of genes with other types of p-element insertion and the  <dig>  kb average size of the genes in the genome without p-element insertions .
the dramatic difference in gene length is mainly due to differences in cumulative intron length.
the mean cumulative intron length for protein trap genes is  <dig>  kb, compared to  <dig>  kb for other genes with other p-element insertions .
two factors contribute to the higher cumulative intron length : protein trap targets have a larger number of introns  and the individual introns are longer  compared to other genes with non-protein trap p-element insertions.
the protein trap insertions preferentially land in longer introns since trapped introns are, on average,  <dig>  kb long, significantly longer than the  <dig>  kb average for other introns within the same genes .
these numbers confirm trends already recognised in the pilot studies by morin et al.
<cit> , where for the candidate gene set the average length of targeted introns was reported larger than  <dig>  kb and thus significantly larger than the genomic average.
this observation provides one explanation for how protein trap targets differ from other genes susceptible to p-element integration since they contain larger landing spaces for the gfp-encoding exon.
it is noteworthy that a similar trend is also observed for introns trapped with piggybac vectors  since the introns hit by piggybac are on average  <dig>  kb long, more than  <dig> times larger than the genome-wide intron length average of  <dig>  kb .
protein trap targets show broad and persistent gene expression
host gene expression has a direct influence on gfp detection in protein trap screens.
detection success largely depends on two parameters: signal strength  and expression duration .
in general, protein trap screens select individual gfp-positive progeny generated in a particular cross from a much larger background of gfp-negative individuals.
the screens generally take a momentary snapshot of the population and thus, in order to be recovered, a tagged protein must be expressed at detectable levels when the selection is made.
morin et al.
screened first instar larvae manually with a dissecting microscope, while both of the large-scale screens utilised an automated embryo sorter.
good examples of the importance of expression timing were observed by quiñones-coello et al.
<cit> .
they found that the dek gene  was independently hit  <dig> times by morin et al.
<cit>  but never in the embryo based screen, and vice versa with extra macrochaetae , which was detected  <dig> times by quiñones-coello et al.
but not by morin et al.
however, even within the period of expression in the embryo, our analysis quantified biases linked to expression.
to assess the contribution of host gene expression to protein trap recovery, spatio-temporal gene expression was analysed, where available, for genes containing p-element protein trap insertions.
for this analysis, drosophila embryonic development was broadly divided into three roughly equivalent time frames ; ii = stage 9– <dig>  4– <dig> h ael; iii = stage 13– <dig>  9– <dig> h ael).
using data from the bdgp in situ database  <cit> , we determined in how many of these three time frames each target gene is expressed.
expression information was available for  <dig> out of the  <dig> protein-trapped genes.
the set of gfp-tagged genes is significantly biased towards expression in two or three time frames , whereas only slightly more than 50% of other genes with non-trap p-element insertions show prolonged expression .
this is expected, since the longer a gene is expressed the greater the likelihood that it will be detected in a screen.
the expression of successfully tagged genes is not only persistent, but also tends to occur in broad domains and comparatively large tissues.
a striking  <dig> of the  <dig> genes  with expression information are annotated as being maternally contributed where, in most cases, the tagged protein is expected to be distributed throughout the embryo.
this is an interesting observation because in both large-scale screens, p-elements were mobilised in males and thus the initial selection could not be based on maternal contributions.
many tagged genes also exhibit ubiquitous expression in later embryonic stages .
other relatively large tissues that are enriched for protein-trapped genes include the embryonic brain , the ventral nerve cord  and various tissues of mesodermal origin.
this suggests that, if the gfp signal follows mrna expression, most gfp-tagged genes are expressed in at least 20% of the total embryonic cell population.
genes tagged using a piggybac vector display the same trends.
although expression data is available for only  <dig> of the  <dig> tagged genes, these tend to be broadly expressed, on average in  <dig>  of the  <dig> stages.
the spatial and temporal data taken together confirm the assumption that there is a detection bias for genes with persistent and broad embryonic expression.
introns at exon-exon boundaries that map to unstructured protein regions are frequently hit
a functional target protein can only be obtained if integration of the  <dig> kda gfp polypeptide occurs in places where it is not structurally constrained.
in cases where gfp insertion into a protein disrupts the folding or is otherwise detrimental, we expect that the tagged protein will be degraded and thus such lines are unlikely to be recovered in screens.
in fact, 36% of the canonical protein trap lines studied by quiñones-coello et al.
<cit>  were homozygous lethal, of which two thirds were associated with the gfp-exon by genetic complementation experiments.
in general, therefore, the recovered protein trap lines provide a snapshot of insertions that are tolerated with respect to protein structure and folding.
the crystal structure of gfp indicates that its n- and c-termini are in relatively close proximity  <cit> , suggesting that gfp may be able to act as a linker between individual protein domains or can be inserted at surface exposed areas within domains without altering the overall fold of the targeted protein.
we classified the  <dig>  protein regions encoded around exon-exon boundaries in the gfp-tagged transcripts, of which  <dig> are interrupted by the gfp insertion.
we found a statistically significant preference  for structurally disordered/unfolded regions as predicted by disopred <dig>  <cit>  , whereas unclassifiable regions are hit at similar rate .
domains predicted in the pfam protein families database  <cit> , excluding those overlapping with disordered regions, appear to be avoided .
interestingly, manual inspection of three-dimensional structures of directly hit pfam domains showed that the gfp insertion sites are located mostly at the surface, leaving the overall fold of the targeted domain unchanged .
in the example of rho1-gfp, the gfp insertion  into the ras domain  takes place at a solvent exposed β-strand  distant from the gtp binding site.
sar <dig> is a protein of the arf family and the example structure shows the small gtp binding protein arf <dig>  in complex with its activator, gef-arno .
sequence mapping places the gfp insertion in a solvent exposed loop remote from both nucleotide and activator binding sites.
in both examples, the active domains of the targets in question remain unaffected by the gfp insertion.
in the course of this analysis we also identified a variety of correlations between intron features, as well as between intron features and structural features encoded at the respective exon-exon boundaries .
for example, the mean length of introns at unstructured boundaries is approximately  <dig>  kb, more than twice as long as the mean of those interrupting pfam domains .
the preference of p-element insertions for longer introns suggests that the identified bias for disordered/unfolded regions may be a result of the general insertion bias, and not of detrimental effects to protein structure.
while neither of these two possibilities can be ruled out, we observed that the  <dig> introns at exon-exon boundaries in gfp-tagged genes that do not have a protein trap insertion but do have a non-trap insertion show essentially the same characteristics as those without any transposon insertion .
this observation suggests that insertion bias alone is not sufficient to explain the gfp trap preference for unstructured regions.
it is noteworthy that protein-trapped genes often exhibit a number of splice variants, some of which are not affected by the trap insertion: of  <dig> gfp-tagged genes,  <dig>  are predicted to have additional unaffected transcripts.
it should be noted that the impact of gfp insertion on protein structure cannot fully be assessed due to the lack of experimental data and because only 20% of the primary protein sequence in the fly possesses reliable structural predictions .
the majority of likely targets have already been hit
morin  <cit>  estimated that only a couple of hundred loci are likely to be successfully targeted by p-elements, and that different vectors will soon be required.
based on our in-depth analysis from the data that have since become available, we set out to more precisely quantify this prediction.
taking the above observations together we identified insertion biases, gene expression and protein structural constraints as likely limiting factors for the success of protein trap screens.
a cumulative score representing the likelihood of a successful insertion was calculated independently for each intron.
the parameters used for the prediction included the number of previous p-element hits, the length of the intron, the presence of intrinsic disorder in the protein region encoded around the exon-exon boundary, and, where available, the expression data for the gene in question .
the model was used to generate a list of high probability candidate genes from the set of all genes with expression information.
we then asked, how many of the top-ranked genes from this list need to be accepted to recover the p-element-based protein trap insertions listed in flytrap for which gene expression data is available .
for example, to identify 80% of the genes in flytrap we need to accept  <dig> predicted targets.
of these  <dig> genes, only 34% have been identified in protein trap screens.
analogously, the analysis was repeated for genes with no expression information considering only the other three model parameters.
in order to identify 80% of the flytrap genes with unknown expression,  <dig> genes of unknown expression need to be accepted .
taken together,  <dig> high-ranking genes recover 80% of the p-element-based gfp-tagged genes .
we also applied our estimation to data from a set of piggybac-based protein traps, combining the  <dig> lines from flytrap with  <dig> lines from an ongoing screen at the university of cambridge .
under the assumption that similar biases apply to piggybac-based protein trap screens, we predict more than  <dig>  potential target genes to recover 80% of lines already identified .
this suggests that there is a larger set of genes still amenable to protein-tagging using piggybac compared with p-element vectors.
the respective overlap between previously identified gfp-tagged genes with estimations based on 80% recovery for both transposable elements  is shown in figure 4c.
this indicates that p-elements are reaching the end of their useful life as protein trap vectors, whereas screens utilising piggybac are likely to still have potential for yielding more trapped genes.
however, the analysis also suggests that the utility of piggybac vectors is also finite and other methods will have to be developed to achieve genome-wide coverage.
CONCLUSIONS
transposable element-based protein trap screens have strong underlying biases, largely dependent on which transposon vector is used.
while p-element based screens are predicted to yield additional new inserts, this is likely to be at very low efficiency, since most high-probability targets have already been hit.
studies employing piggybac-based vectors appear to be more promising, with potentially  <dig> times as many targets expected compared with the p-element-based approach.
however, given the significant overlap between the predicted target genes obtainable with both vectors and the existence of restraints on their insertion location, transposable element-based approaches are likely to reach the end of their utility in the near future.
an assessment of the possibility of using a different transposon vector such as minos, which exhibits a more random insertion pattern  <cit> , is therefore recommended.
at present there are insufficient data for us to predict the number of minos targets in the drosophila genome.
in the long run, to achieve true genome-wide coverage or to target functionally important gene classes such as transcription factors, targeting vectors utilising homologous recombination will have to be designed for protein trapping.
structural restraints will still apply in such cases and we note that optimal exon-exon boundaries encoding regions with local disorder are only predicted for approximately half of the intron containing genes in drosophila.
the use of homologous recombination methods combined with careful construct design, taking into account the underlying limitations of protein trap screens, could be a potential route for attaining better genome coverage.
alternatively, a recombineering-based approach in drosophila has recently been developed  <cit> , which allows tagging of specific genes with a variety of functional moieties, and is amenable to high-throughput applications.
while protein tagging recombineering studies have not yet been reported, it is likely that this method may well lead to more complete genome coverage.

 genetic trapping experiments have a long-standing history in drosophila functional genomics. the classic "enhancer trap" screens utilised p-element-mediated insertion of the e. coli lacz gene to facilitate relatively unbiased discovery of tissue-specifically expressed genes and enhancers at a genome-wide level. in recent years, additional transposable elements such as piggybac or minos have found their way into research applications. together, this set of transposons provides excellent tools for genome-wide genetic screens. while p-elements and piggybac show some positional preferences towards insertion at the 5' regions of target genes, and thus have biased coverage with respect to the genome, minos is reported to show a much more random genomic distribution of insertions and may be more suited to genome-wide screens. the introduction of the green fluorescent protein in research applications opened new avenues for enhancer trapping, since the expression of a fluorescent reporter can be directly observed and detection does not require an enzymatic reaction. a more recent advance in the gene trapping approach is the "protein trap" screen, which aims to create gfp-tagged versions of endogenous proteins under the control of the genes' native regulatory sequences. as is evident from the comprehensive recombination-based efforts in yeast, tagging endogenous proteins in vivo can have tremendous utility for genomics, cell biology and systems biology studies. protein tagging is likely to be of even greater untility in metazoans where many different cell types are present. protein trap screens utilise artificial reporter-encoding exons to generate fluorescent fusion proteins by random integration into the genome. the reporter is usually a gfp variant, flanked by splice acceptor/donor sites, and carried within a transposable element vector. integration of the transposon within an intron in the correct orientation results in the transcription and subsequent splicing of the trapping exon into the mature mrna of the targeted gene. if the trapping exon is in-frame with coding sequence of the host protein, a functional gfp-tagged version of the protein may be produced. a comprehensive screen obviously requires vectors carrying targeting exons in all three reading frames, but even when multiple vectors are employed the isolation of bona fide protein traps is a relatively rare event. following the pioneering work of morin et. al., the results from a variety of drosophila protein trap screens have been published. in total, these studies have screened close to million individual embryos or larvae, with only a small fraction of these generating lines that express the gfp tag. the flytrap database reports fluorescent lines generated from the three largest protein trap screens performed to date. mapping of their insertion coordinates to release gene annotation shows that these represent unique genes tagged with protein traps located within introns separating coding exons. with the fly genome containing approximately protein-coding genes, the screens have hit less than 2% of known fly genes. this is far from genome-wide coverage, clearly a desirable goal for comprehensive functional genomics studies. the restricted success of protein trap screens is especially surprising given that approximately drosophila genes contain introns. thus, in principle, approximately 80% of fly genes are accessible to protein trapping. interestingly, although the overall number of unique lines generated in the different screens is relatively small, there is considerable overlap in the tagged genes recovered by the individual screens. the low efficiency and high degree of overlap between the published screens suggests that there are limitations to the protein trap strategies currently in use. here we attempt to identify and quantify these limitations, and suggest future strategies that may increase the repertoire of trapped genes. we considered a number of potential factors that could bias protein trap screens, including transposable element integration hotspots, gene architecture, gene expression and protein structure. we constructed a probability model that we used to predict a set of target genes with a high likelihood of successfully receiving a protein trap insertion. our model predicts that approximately of the genes encoded in the fly genome are permissive for p-element based protein trapping and of these, genes have already been tagged in previous studies. a similar analysis based on data from a more limited set of protein traps generated with piggybac vectors estimates approximately genes are permissive targets with this transposon, and about of these have not yet been tagged in previous studies. comparing the predictions for both transposons we find that most potential as yet untagged p-element targets are also good piggybac targets. due to the apparent importance of transposon insertion bias, it is likely that a transposable element such as minos, which exhibits a more random insertion preference, may be a better vector for future random protein trap screens. ultimately, it is likely that recombination or recombineering -based gene targeting techniques will need to be employed to achieve comprehensive coverage of the fly genome.  the minority of gfp trap lines are real protein traps we retrieved insertion data for previously described protein trap lines from the flytrap database. the genomic insertion coordinates were available for of the lines and these were taken forward for further analysis. the insertion sites from flytrap were translated to drosophila genome release coordinates and mapped with respect to the release gene annotations taken from flybase. the exon boundary and coding sequence information was obtained for each transcript of the relevant genes and, where available, the relevant frame information from the original protein trap screen was utilised. together, these data established a catalogue of protein trap target genes based on the most recent genome sequence and annotation releases. the compiled data are available with a summary provided in table the updated insertion coordinates were mapped to genes from flybase release, and the insertion annotations were updated accordingly. the mapping of a small number of lines was ambiguous due to the insertion being in various features of different transcripts and/or different nested genes – in the cases where an intron between coding sequence was hit, the line was classified as'cds intron', regardless of the other features hit concurrently. some genes are represented by multiple lines, so the fly lines hitting cds introns in fact amount to unique genes hit. a canonical protein trap is defined as an insertion residing in an intron within the coding sequence of a gene that, after splicing, allows the translation of the gfp reporter in frame with the endogenous polypeptide. the recovery of enhancer traps in protein trap screens has been previously observed and determined to be due to the presence of an upstream start codon. the start codon is within the transposase gene of the protein trap vector itself, but has also been reported to result from a splicing event from the non-coding strand of the mini-white marker present in the vector. the updated release coordinates and gene annotations confirm that bona fide protein traps constitute the minority of the reported lines. of the fly lines for which coordinates were available, are insertions entirely outside gene regions and of the remaining lines that do insert within a gene model, only are found in introns between coding exons. these insertions tag a total of unique genes, reducing to genes if frame information is taken into account. the insertions in places other than the introns within coding sequence are considered to be enhancer trap type insertions rather than protein traps. of the flytrap lines have insertions targeting gene regions outside their coding sequence. interestingly, only of such insertions are located in the 3' utr of genes with the remainder 5' to the gene start codon. while this suggests that it is indeed likely that the p-element promoter and translational start sites facilitate enhancer trapping, some of the traps may be located in introns associated with unannotated 5' exons and thus may represent bona fide protein traps. insertion biases of protein trap insertions approximately protein-coding genes in the fly genome have at least one intron and therefore 80% of the drosophila gene repertoire is in principle amenable to gfp-tagging. in practise, the fraction of recovered protein traps is clearly much lower, indicating that there are preferred target genes. this becomes evident when the total of gfp-tagged proteins found in the three published large-scale screens are compared. while the largest screen successfully tagged a number of new proteins, most of the proteins tagged in the smaller screens are recovered in multiple studies. 68% of the protein traps reported by morin et al. and 66% of those reported by quiñones-coello et al. were also recovered in one or both of the other screens. transposable element hotspots introduce the greatest bias in protein trap screens the p-element constructs of morin et al. were used in the two other screens reported in flytrap and lines derived with these vectors represent the majority of the insertions reported in the database. the remaining insertions were generated with a piggybac vector. to explore the possibility that p-element insertion preference biases the recovery of protein traps, we compared the repertoire of genes recovered in the protein trap screens with all of the p-element insertions reported in flybase. twelve genes were independently recovered using p-elements in all three protein trap screens and we found an average of other p-element insertions in each of these genes. these genes alone are covered by independent lines reported in flytrap, accounting for % of all the lines in the database and over 13% of coding sequence insertions. this observation suggests that there may be significant bias due to p-element insertion hotspots. noteworthy, amongst the targets shared between all protein trap studies is cg which accounts for 10% of all "kg" lines recovered in the gene disruption project. for all remaining protein trap targets generated using p-element vectors, the insertion location is strongly biased. of the genes containing p-element gfp trap insertions into coding sequence introns, have other p-element insertions and only genes have no previously reported transposon insertions. the frequency of p-element insertions in genes isolated in the protein trap screens is significantly higher than observed within the approximately protein-coding genes that have reported p-element integrations. in addition, within the protein trap tagged genes, the integration hotspots are localised within the tagged intron since these have, on average, other p-element insertions. other introns within tagged genes have, on average, only p-element insertions. two of the published protein trap screens also used piggybac-based vectors, because of their reported preference for at-rich integration sites such as introns. these pilot screens generated lines of which insertions result in protein traps in unique genes. interestingly, the same bias towards previously isolated transposon targets is also found in the piggybac set with out of genes containing previously reported transposon insertions: of the have other piggybac insertions. a total of previously reported piggybac insertions are associated with the protein trapped genes recovered in the piggybac screens: a comparable level to that observed in p-element screens. the high frequency of other transposable element insertions associated with genes recovered in the protein trap screens indicates that these genes, and more specifically their tagged introns, are genomic hotspots for transposon insertion. gfp traps selectively target longer introns due to the much larger number of p-element protein trap lines reported in flytrap, we concentrate the majority of our further analysis on the differences between genes generally susceptible to p-element integration and genes recovered in the protein trap screens, to identify factors that may influence recovery of protein traps. the average length of the genes successfully tagged by p-element protein traps is kb, significantly higher than the kb average size of genes with other types of p-element insertion and the kb average size of the genes in the genome without p-element insertions. the dramatic difference in gene length is mainly due to differences in cumulative intron length. the mean cumulative intron length for protein trap genes is kb, compared to kb for other genes with other p-element insertions. two factors contribute to the higher cumulative intron length: protein trap targets have a larger number of introns and the individual introns are longer compared to other genes with non-protein trap p-element insertions. the protein trap insertions preferentially land in longer introns since trapped introns are, on average, kb long, significantly longer than the kb average for other introns within the same genes. these numbers confirm trends already recognised in the pilot studies by morin et al., where for the candidate gene set the average length of targeted introns was reported larger than kb and thus significantly larger than the genomic average. this observation provides one explanation for how protein trap targets differ from other genes susceptible to p-element integration since they contain larger landing spaces for the gfp-encoding exon. it is noteworthy that a similar trend is also observed for introns trapped with piggybac vectors since the introns hit by piggybac are on average kb long, more than times larger than the genome-wide intron length average of kb. protein trap targets show broad and persistent gene expression host gene expression has a direct influence on gfp detection in protein trap screens. detection success largely depends on two parameters: signal strength and expression duration. in general, protein trap screens select individual gfp-positive progeny generated in a particular cross from a much larger background of gfp-negative individuals. the screens generally take a momentary snapshot of the population and thus, in order to be recovered, a tagged protein must be expressed at detectable levels when the selection is made. morin et al. screened first instar larvae manually with a dissecting microscope, while both of the large-scale screens utilised an automated embryo sorter. good examples of the importance of expression timing were observed by quiñones-coello et al.. they found that the dek gene was independently hit times by morin et al. but never in the embryo based screen, and vice versa with extra macrochaetae, which was detected times by quiñones-coello et al. but not by morin et al. however, even within the period of expression in the embryo, our analysis quantified biases linked to expression. to assess the contribution of host gene expression to protein trap recovery, spatio-temporal gene expression was analysed, where available, for genes containing p-element protein trap insertions. for this analysis, drosophila embryonic development was broadly divided into three roughly equivalent time frames; ii = stage 9– 4– h ael; iii = stage 13– 9– h ael). using data from the bdgp in situ database, we determined in how many of these three time frames each target gene is expressed. expression information was available for out of the protein-trapped genes. the set of gfp-tagged genes is significantly biased towards expression in two or three time frames, whereas only slightly more than 50% of other genes with non-trap p-element insertions show prolonged expression. this is expected, since the longer a gene is expressed the greater the likelihood that it will be detected in a screen. the expression of successfully tagged genes is not only persistent, but also tends to occur in broad domains and comparatively large tissues. a striking of the genes with expression information are annotated as being maternally contributed where, in most cases, the tagged protein is expected to be distributed throughout the embryo. this is an interesting observation because in both large-scale screens, p-elements were mobilised in males and thus the initial selection could not be based on maternal contributions. many tagged genes also exhibit ubiquitous expression in later embryonic stages. other relatively large tissues that are enriched for protein-trapped genes include the embryonic brain, the ventral nerve cord and various tissues of mesodermal origin. this suggests that, if the gfp signal follows mrna expression, most gfp-tagged genes are expressed in at least 20% of the total embryonic cell population. genes tagged using a piggybac vector display the same trends. although expression data is available for only of the tagged genes, these tend to be broadly expressed, on average in of the stages. the spatial and temporal data taken together confirm the assumption that there is a detection bias for genes with persistent and broad embryonic expression. introns at exon-exon boundaries that map to unstructured protein regions are frequently hit a functional target protein can only be obtained if integration of the kda gfp polypeptide occurs in places where it is not structurally constrained. in cases where gfp insertion into a protein disrupts the folding or is otherwise detrimental, we expect that the tagged protein will be degraded and thus such lines are unlikely to be recovered in screens. in fact, 36% of the canonical protein trap lines studied by quiñones-coello et al. were homozygous lethal, of which two thirds were associated with the gfp-exon by genetic complementation experiments. in general, therefore, the recovered protein trap lines provide a snapshot of insertions that are tolerated with respect to protein structure and folding. the crystal structure of gfp indicates that its n- and c-termini are in relatively close proximity, suggesting that gfp may be able to act as a linker between individual protein domains or can be inserted at surface exposed areas within domains without altering the overall fold of the targeted protein. we classified the protein regions encoded around exon-exon boundaries in the gfp-tagged transcripts, of which are interrupted by the gfp insertion. we found a statistically significant preference for structurally disordered/unfolded regions as predicted by disopred, whereas unclassifiable regions are hit at similar rate. domains predicted in the pfam protein families database, excluding those overlapping with disordered regions, appear to be avoided. interestingly, manual inspection of three-dimensional structures of directly hit pfam domains showed that the gfp insertion sites are located mostly at the surface, leaving the overall fold of the targeted domain unchanged. in the example of rho1-gfp, the gfp insertion into the ras domain takes place at a solvent exposed β-strand distant from the gtp binding site. sar is a protein of the arf family and the example structure shows the small gtp binding protein arf in complex with its activator, gef-arno. sequence mapping places the gfp insertion in a solvent exposed loop remote from both nucleotide and activator binding sites. in both examples, the active domains of the targets in question remain unaffected by the gfp insertion. in the course of this analysis we also identified a variety of correlations between intron features, as well as between intron features and structural features encoded at the respective exon-exon boundaries. for example, the mean length of introns at unstructured boundaries is approximately kb, more than twice as long as the mean of those interrupting pfam domains. the preference of p-element insertions for longer introns suggests that the identified bias for disordered/unfolded regions may be a result of the general insertion bias, and not of detrimental effects to protein structure. while neither of these two possibilities can be ruled out, we observed that the introns at exon-exon boundaries in gfp-tagged genes that do not have a protein trap insertion but do have a non-trap insertion show essentially the same characteristics as those without any transposon insertion. this observation suggests that insertion bias alone is not sufficient to explain the gfp trap preference for unstructured regions. it is noteworthy that protein-trapped genes often exhibit a number of splice variants, some of which are not affected by the trap insertion: of gfp-tagged genes, are predicted to have additional unaffected transcripts. it should be noted that the impact of gfp insertion on protein structure cannot fully be assessed due to the lack of experimental data and because only 20% of the primary protein sequence in the fly possesses reliable structural predictions. the majority of likely targets have already been hit morin estimated that only a couple of hundred loci are likely to be successfully targeted by p-elements, and that different vectors will soon be required. based on our in-depth analysis from the data that have since become available, we set out to more precisely quantify this prediction. taking the above observations together we identified insertion biases, gene expression and protein structural constraints as likely limiting factors for the success of protein trap screens. a cumulative score representing the likelihood of a successful insertion was calculated independently for each intron. the parameters used for the prediction included the number of previous p-element hits, the length of the intron, the presence of intrinsic disorder in the protein region encoded around the exon-exon boundary, and, where available, the expression data for the gene in question. the model was used to generate a list of high probability candidate genes from the set of all genes with expression information. we then asked, how many of the top-ranked genes from this list need to be accepted to recover the p-element-based protein trap insertions listed in flytrap for which gene expression data is available. for example, to identify 80% of the genes in flytrap we need to accept predicted targets. of these genes, only 34% have been identified in protein trap screens. analogously, the analysis was repeated for genes with no expression information considering only the other three model parameters. in order to identify 80% of the flytrap genes with unknown expression, genes of unknown expression need to be accepted. taken together, high-ranking genes recover 80% of the p-element-based gfp-tagged genes. we also applied our estimation to data from a set of piggybac-based protein traps, combining the lines from flytrap with lines from an ongoing screen at the university of cambridge. under the assumption that similar biases apply to piggybac-based protein trap screens, we predict more than potential target genes to recover 80% of lines already identified. this suggests that there is a larger set of genes still amenable to protein-tagging using piggybac compared with p-element vectors. the respective overlap between previously identified gfp-tagged genes with estimations based on 80% recovery for both transposable elements is shown in figure 4c. this indicates that p-elements are reaching the end of their useful life as protein trap vectors, whereas screens utilising piggybac are likely to still have potential for yielding more trapped genes. however, the analysis also suggests that the utility of piggybac vectors is also finite and other methods will have to be developed to achieve genome-wide coverage.  transposable element-based protein trap screens have strong underlying biases, largely dependent on which transposon vector is used. while p-element based screens are predicted to yield additional new inserts, this is likely to be at very low efficiency, since most high-probability targets have already been hit. studies employing piggybac-based vectors appear to be more promising, with potentially times as many targets expected compared with the p-element-based approach. however, given the significant overlap between the predicted target genes obtainable with both vectors and the existence of restraints on their insertion location, transposable element-based approaches are likely to reach the end of their utility in the near future. an assessment of the possibility of using a different transposon vector such as minos, which exhibits a more random insertion pattern, is therefore recommended. at present there are insufficient data for us to predict the number of minos targets in the drosophila genome. in the long run, to achieve true genome-wide coverage or to target functionally important gene classes such as transcription factors, targeting vectors utilising homologous recombination will have to be designed for protein trapping. structural restraints will still apply in such cases and we note that optimal exon-exon boundaries encoding regions with local disorder are only predicted for approximately half of the intron containing genes in drosophila. the use of homologous recombination methods combined with careful construct design, taking into account the underlying limitations of protein trap screens, could be a potential route for attaining better genome coverage. alternatively, a recombineering-based approach in drosophila has recently been developed, which allows tagging of specific genes with a variety of functional moieties, and is amenable to high-throughput applications. while protein tagging recombineering studies have not yet been reported, it is likely that this method may well lead to more complete genome coverage. 
despite recent efforts aimed at achieving comprehensive coverage of the genes encoded in the drosophila genome, the repertoire of genes that yield protein traps is still small. in addition, we investigated the likely influence of the gfp exon on host protein structure and found that protein trap insertions have a significant bias for exon-exon boundaries that encode disordered protein regions. interestingly, even in cases where protein domains are predicted, protein trap insertions frequently occur in regions encoding surface exposed areas that are likely to be functionally neutral. our analyses suggest that the utility of p-element vectors for protein trap screens has largely been exhausted, and that approximately genes may still be amenable using piggybac vectors. the ability to localise or follow endogenous proteins in real time in vivo is of tremendous utility for cell biology or systems biology studies. the protein trap screens investigated here primarily used p-element vectors and thus exhibit some of the same positional biases associated with this transposon that are evident from the comprehensive drosophila gene disruption project. we analysed the collection of available protein trap lines in drosophila melanogaster and identified potential biases that are likely to restrict genome coverage in protein trap screens. we suggest that either transposons with reduced insertion bias or recombineering-based targeting techniques will be required for comprehensive genome coverage in drosophila. protein trap screens utilise the random genomic insertion of a transposon-borne artificial reporter exon into an intron of an endogenous gene to generate a fluorescent fusion protein. we further found that protein trap target genes usually exhibit broad and persistent expression during embryonic development, which is likely to facilitate better detection. % of gfp insertions land in disordered protein regions compared with only % in the case of non-trapping p-element insertions landing in coding sequence introns. thus protein trap strategies based on current approaches are unlikely to offer true genome-wide coverage.
 the ability to localise or follow endogenous proteins in real time in vivo is of tremendous utility for cell biology or systems biology studies. protein trap screens utilise the random genomic insertion of a transposon-borne artificial reporter exon into an intron of an endogenous gene to generate a fluorescent fusion protein. despite recent efforts aimed at achieving comprehensive coverage of the genes encoded in the drosophila genome, the repertoire of genes that yield protein traps is still small.  we analysed the collection of available protein trap lines in drosophila melanogaster and identified potential biases that are likely to restrict genome coverage in protein trap screens. the protein trap screens investigated here primarily used p-element vectors and thus exhibit some of the same positional biases associated with this transposon that are evident from the comprehensive drosophila gene disruption project. we further found that protein trap target genes usually exhibit broad and persistent expression during embryonic development, which is likely to facilitate better detection. in addition, we investigated the likely influence of the gfp exon on host protein structure and found that protein trap insertions have a significant bias for exon-exon boundaries that encode disordered protein regions. % of gfp insertions land in disordered protein regions compared with only % in the case of non-trapping p-element insertions landing in coding sequence introns. interestingly, even in cases where protein domains are predicted, protein trap insertions frequently occur in regions encoding surface exposed areas that are likely to be functionally neutral. considering the various biases observed, we predict that less than one third of intron-containing genes are likely to be amenable to trapping by the existing methods.  our analyses suggest that the utility of p-element vectors for protein trap screens has largely been exhausted, and that approximately genes may still be amenable using piggybac vectors. thus protein trap strategies based on current approaches are unlikely to offer true genome-wide coverage. we suggest that either transposons with reduced insertion bias or recombineering-based targeting techniques will be required for comprehensive genome coverage in drosophila. 