One of the many advantages of Drosophila melanogaster as a model organism is the relative ease with which gene deletions can be generated by imprecise excision of transposon insertions. Here, we describe a simple, fast, and efficient method of screening for single-gene excision events that is not biased by prior assumptions of the mutant phenotype. DNA sequence polymorphisms were used as co-dominant electrophoretic markers to identify candidate deletions in a single generation, and to delimit the breakpoints to within 0.5–1 kb, thereby rapidly identifying deficiencies that affect only the gene of interest. In addition, we used polymorphism profiling to map existing deficiencies. The method can also be applied to map the extent of deletions generated by x-rays and to identify targeted mutations generated by engineered zinc-finger nucleases in Drosophila and other polymorphic model organisms (e.g., zebrafish, mouse, Caenorhabditis elegans).

Introduction

Reverse genetics is a powerful approach for studying gene function in vivo. In Drosophila, a good model for studying eukaryotic gene function because of the wide range of available mutations and the relative lack of genetic redundancy, there are many different ways to selectively disrupt the function of genes of interest (1,2). These include ends-out replacement gene targeting by homologous recombination (3), post-transcriptional knockdown via dsRNA (4), and zinc-finger nuclease–induced targeting (5,6), or by using site-directed recombination between FRT sites in trans (7,8). Perhaps the most straight forward method is imprecise excision of transposable elements inserted adjacent to the target gene (9). To date, transposon insertions have been identified for more than two-thirds of annotated genes in D. melanogaster, and this proportion is ever increasing due to several genome-wide transposon mutagenesis projects (10,11).

Drosophila has several different types of transposable elements (including P, minos, and piggyBac) (7,11,12) whose transposases catalyze transposon excision by generating a double-strand break in the genomic DNA. Although this break is usually repaired precisely, the DNA break ends for P and minos elements can be rejoined via non-homologous end joining (NHEJ), which causes deletions at the break site (13) that can be exploited to generate localized deficiencies.

The frequency of such imprecise events varies from locus to locus (1 in 5 to 1 in 100 excision events) (1,11), and the frequency of imprecise excision can be increased dramatically in some specific genetic backgrounds or by inhibiting pairing between the transposable element chromosome and its homolog (14,15). Flies carrying a visibly marked [e.g., white⁺ (w ⁺)] transposable element inserted into or near a gene of interest are crossed with flies containing a stable source of transposase. Flies in which the element has been excised are identified by loss of the visible marker (e.g., w ⁻) and can be tested individually for predicted or expected homozygous or hemizygous phenotypes.

The alternative to phenotypic assays is to identify imperfect excision events molecularly. This can be done by Southern blotting; however, this method is unsuitable for large-scale screening, and also misses deletions that remove the DNA probe region. Another technique is to use PCR on genomic DNA from heterozygous flies with flanking primer sites that are too distant to amplify a wild-type or trans-poson-containing fragment, but that would amplify a fragment made smaller by a suitable deletion. Such PCR screens are often inefficient because the amplified fragment—whose size cannot be predicted in advance—might be poorly resolved or visualized on the chosen gel system. The method can also fail if too large a footprint of the transposable element is still present on the chromosome, or if there are small but inactivating deletions.

Alternatively, one can use PCR on genomic DNA from homozygous flies, and screen for the loss of a genomic fragment. This method is efficient but relies on the homozygous mutants being viable, and requires extra generations to produce homozygous flies. Finally, quantitative, real-time PCR can be used to detect the reduced gene copy number (16), but it is technically challenging to screen a large number of flies for a two-fold difference in gene dosage.

Here, we report a simple, rapid, and efficient strategy to screen and map the extent of transposon-induced deletions of any locus of interest in a single generation using polymorphic sequence markers. This method, which makes no assumptions about the mutant phenotype and uses pretested primers, is based on the comparison of polymorphic sequences between the strain carrying the transposable element and an unrelated tester strain, and identifies all deletions in the polymorphic region. Imprecise excision events are revealed by selective loss of polymorphic alleles associated with the strain bearing the transposable element. All steps including the screening and the mapping can be achieved within a single generation, substantially reducing the time and stock maintenance required. We have validated this method by identifying single-gene deletions at two different loci in the Drosophila genome. This method can also be used to map deletions generated in other ways (including via x-rays), or adapted for use with other polymorphic genetic model organisms.

Materials and methods

Drosophila stocks

All Drosophila stocks and crosses were set up on standard medium at 25°C. The w;MKRS, P{Δ2–3}/TM6B, Tb, w;TM6B,Tb/Sb, w;P{EPgy2}HUS1-like^EY14510, w;Df(3R)ED₅₀95/TM6C, cu, Sb (Deficiency for 82C5;82E7), Df(3L) Cat (75C1–75C2;75F1), and y¹, w^67c23; P{lacW}RpL11^k16914/CyO lines were obtained from the Bloomington Stock Center (Indiana University, Bloomington, IN, USA). P{GawB}^NP3550 was provided by the Drosophila Genetic Resource Center (DGRC; Kyoto Institute of Technology, Kyoto, Japan). We used isogenized parental chromosomes, although intrapopulation polymorphisms are relatively uncommon in inbred stocks predicted to be monomorphic (17).