A complementary transposon tool kit for drosophila melanogaster

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The P-element transposon, the workhorse of D. We describe specific improvements to the… Expand. View on Nature. Save to Library Save. Create Alert Alert. Share This Paper. Background Citations. Methods Citations.

Results Citations. Figures, Tables, and Topics from this paper. Citation Type. We incorporated three technological improvements to increase the efficiency of transposon mutagenesis in D.

First, we used an additional mobile genetic element. Alternative transposons have been used in D. We selected piggyBac as the most promising transposon for this application. Third, although previous screens typically mobilized transposons in the male germ line, differences in transposon activity between male and female germ lines 8 led us to use the female germ line, with the hope of altering the spectrum of genes accessible to tagging.

An ongoing challenge for fruit fly researchers is that mutants and transgenic strains come from a variety of genetic backgrounds that may harbor pre-existing mutations 9 , thus precluding or complicating highly sensitized or behavioral screens. We generated all our transposon reagents and associated stocks in a freshly derived isogenic background; the insertion collection reported here is fully isogenic. Molecular and genetic analysis suggests that this was not simply due to multiple insertions.

Eighty-nine percent of the lines yielded unique flanking sequence by inverse PCR. In addition, 97 of piggyBac lines analyzed by genomic Southern blotting had single insertions data not shown. The increased lethal frequency observed for piggyBac was not the result of elevated background mutagenic effects 'hit and runs' at sites independent of the final insertion.

Because we started with a clean isogenic strain, we were able to assess the background mutagenic rate with high accuracy using the following tests. We selected ten lethal piggyBac lines and confirmed that each did not complement a corresponding deficiency Table 2 , indicating these mutations are at least closely linked to the insert. We then used six of these lines to generate 55 excision alleles; each was molecularly confirmed as a precise event that also reverted the chromosome to a viable phenotype Table 2.

Previous studies also found a majority of molecularly precise excision events, although lethal alleles generated in a nonisogenic background were not always reverted. Additionally, separate genetic screens using our collection found that only 0. Finally, secondary mutations may arise if transposase expression activates cryptic elements or induces genomic instability. The insertion and transposase strains have been stable for 4 years with no observed breakdown. Taken together, these data support the conclusion that piggyBac acts as an effective mutagen and that the mutations observed are directly caused by the transposon insertion.

The higher frequency of lethal mutations generated by piggyBac versus P cannot be explained by secondary mutations but must result from some other property of the transposon, such as its insertion pattern or the ability of the transposon itself to locally perturb gene function.

To investigate these possibilities, we compared the local and global patterns of piggyBac and XP insertions. We also analyzed an unbiased public set of P insertions EP element 11 for comparison. We found that a larger fraction of piggyBac elements inserted after the transcriptional start site The XP collection was also less biased than EP by this metric, possibly because we used female dysgenesis in our screen. Excluding first exons, piggyBac tagged remaining exons more than three times more frequently.

Because initial exons in D. Intronic insertions may also be disruptive by interfering with regulatory sequences or proper gene splicing 13 , Thus, because piggyBac insertions occur more frequently between transcriptional start and stop, they should create null alleles more commonly.

Examining molecular placements identified hot-spot regions for both XP and piggyBac Fig. Defining a hot spot as a kb interval containing 30 or more insertions, we found 23 XP hot spots Supplementary Table 1 online. In contrast, from more than twice as many piggyBac insertions, we found only 26 piggyBac hot spots Supplementary Table 1 online , none of which overlapped XP hot spots.

The mean number of piggyBac insertions per hot-spot bin 38 was also less than that for XP Neither transposon had a random Poisson distribution Fig. Chromosome sequence was binned into kb intervals and the number of transposons in each bin is plotted.

Hot-spot and cold-spot regions are apparent for both transposons. All hot spots are nonoverlapping between P and piggyBac. Results were similar across remaining chromosomes. Overlaid on each histogram is a line representing the predicted Poisson distribution for 8, XP and 17, piggyBac inserts transposons placed on unassembled Chromosome U are excluded from this analysis.

The number of bins containing 30 or more transposons is highlighted in the inset histograms. XP and piggyBac elements had comparable numbers of hot spots, although more than twice as many piggyBac inserts were generated.

To assess relative gene tagging frequencies for P and piggyBac , we plotted the cumulative number of DGC genes tagged against recovery of new insertions Fig. The EP 11 set is included for comparison.

Percentage of DGC-tagged genes was plotted by random sampling of the set with increasing numbers of transposons. The initial gene tagging rate is highlighted in the inset graph. After generating and characterizing thousands of piggyBac and P -element insertions, we find piggyBac to be an efficient and practical gene tagging system in D. These reagents and the associated transposon tool kit will be a useful complement to existing D.

In fact, this collection has already been extensively used by us and our collaborators for biological analysis of gene function in pharmaceutically relevant disease pathways 15 , We used three classes of piggyBac vectors and one P element in the screen Supplementary Fig.

The piggyBac vector PB is the simplest, comprising a complete piggyBac transposon with the open reading frame interrupted by the D. Because exons in P elements can disrupt gene function by acting as ectopic splice acceptors 13 , 14 , we designed the RB vector with an exon from the D. In this issue, Parks et al. We remobilized transposase sources onto the CyO balancer chromosome and characterized them for activity before selecting the isolates used in our screen.

We constructed the inducible piggyBac transposase by placing the piggyBac coding sequence under control of the D. We constructed the constitutive piggyBac transposase by cloning the piggyBac open reading frame under control of the D.

We remobilized the PB element using HsppiggyBac transposase from a single ammunition element on either the X or third chromosome. We outcrossed the resulting dysgenic males to an isogenic w strain. We remobilized the RB element from a single X chromosome insertion in dysgenic males and the WH element from a single ammunition element on the Binsinscy balancer chromosome in dysgenic females.

We outcrossed dysgenic males or virgin females in vials to the isogenic w strain and selected new hops in the following generation. Only a single new insertion was retained per vial. For the P element, XP, we selected an easily mobilized ammunition element among inserts hopped onto the Binsinscy balancer. All lines were mapped to a chromosome by standard genetic methods, examined for homozygous viability and used for recovery of flanking genomic sequence.

Rehm Berkeley Drosophila Genome Project. We purified DNA from five flies per line in well format. We treated products with shrimp alkaline phosphatase and DNA exonuclease I. All primer sequences are available on request.

The sequences recovered from transposon flanks were trimmed for quality and masked of any vector.