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Proc. Natl. Acad. Sci. USA Vol. 91, pp. 719-722, January 1994 Genetics Nature screen: An efficient method for screening natural populations of Drosophila for targeted P-element insertions (transposon tagging/reverse genetics/PCR/mutagenesis) ANDREW G. CLARK*, SCOTT SILVERIAt, WENDY MEYERSt, AND CHARLES H. LANGLEYt *Department of Biology, Pennsylvania State University, University Park, PA 16802; and tCenter for Population Biology, University of California, Davis, CA 95616 Communicated by M. M. Green, October 1, 1993 ABSTRACT The efficiency of molecular techniques is P P TlL TlR T2L T2R making it increasingly necessary to rely on reverse genetics to understand the function ofgenes. Tissue-specific libraries allow one to identify numerous genes that can be cloned, sequenced, P-element Target Target and mapped and whose temporal and tissue-specific pattern of Gene 1 Gene 2 expression are well characterized but whose function remains FIG. 1. Diagram ofthe method by which P elements near a target unknown. In such cases, it is desirable to generate targeted gene are detected. Single oligonucleotides prime DNA synthesis mutations to examine the phenotype ofloss-of-function lesions. within each target gene and from the P-element terminal repeats. TlL Here we describe a method for identifying naturally occurring and TlR are the left and right primers for target gene 1. Target gene variants ofDrosophila melanogaster with specific genes tagged primers are oriented toward each other, so that P-element insertion by a nearby P element. Imprecise P-element excision can then within the gene will be detected. Only if a P element is within about be used to generate a series of small deletions in or near the 2 kb ofa target gene will geometric amplification of a DNA fragment gene. In the method described here, large numbers of wild- result, as indicated by the heavy line. caught males were crossed to balancer females, and inserts were identified in pooled samples by the polymerase chain founded each pooled population until a pure line is isolated. reaction with one primer from each target gene and one primer An alternative approach is to perform the PCR screening on from the P-element terminal repeat. We present the calcula- embryos, retaining females in sequentially smaller batches tions for the probability ofsuccessfully tagging a gene and show until the desired line is identified (3). that it is greatly improved by simultaneously screening inserts As simple and elegant as this method is, it has had only into several genes. If a large natural population is available, a modest success, largely because of the high rate of false nature screen is faster and easier than inducing P-element positives from the PCR. This prompted Hamilton et al. (2) to transposition in the laboratory, but the resulting lines, being suggest an alternative approach in which the P elements that genetically heterogeneous, may require more subsequent work are mobilized have a bacterial plasmid origin of replication to isolate. Using this method to screen the genomes of a10,400 and a carbenicillin-resistance marker. After mobilizing such males, we found P-element inserts in close proximity to 3 of 10 elements in many independent crosses, the elements are genes that were screened. stabilized by genetic removal ofthe A2-3 transposase source. Genomic DNA is prepared from flies pooled across lines, It is frequently desirable to generate mutations that alter or partially digested with EcoRI, and ligated under conditions eliminate the expression of genes for which the only infor- that favor circularization. Competent Escherichia coli cells mation available is the nucleotide sequence. This situation are then transformed with this DNA and selected on carben- arises when genes are cloned from tissue-specific libraries or icillin. The resulting plasmid-rescue library is then labeled by when they are identified by homology with some other gene. nick-translation and used as a hybridization probe to screen Two methods have been published that tag targeted genes in for sequence similarity with a battery of cDNA clones that Drosophila melanogaster by mobilizing transposable P-ele- have been bound to nylon membranes. While this method ments and by employing the polymerase chain reaction (PCR) seems more technically difficult than that of Ballinger and to identify inserts near the gene (1, 2). The first makes use of Benzer (1), the plasmid rescue approach is very efficient for the fact that PCR will amplify a particular sequence even if screening certain large classes ofinserts (e.g., brain cDNAs). it is present in <1% of the genomes from which the template One disadvantage, relevant to the method we report, is the DNA is prepared. The chance that any single P-element requirement for the use of engineered P elements bearing insertion will transpose into the neighborhood of a targeted appropriate markers and plasmid functions. The method of gene is roughly the size of the targeted region divided by the Hamilton et al. (2) is very labor intensive and appears best genome size, a figure on the order of 1/100,000. To avoid suited for the situation in which one does not require an having to perform astronomical numbers of PCRs, Ballinger insertion in a particular gene, but where insertions in any of and Benzer (1) pooled the genomic DNA isolated from 100 a large class of genes are informative and useful. males with independent P-element insertions. PCR with one The key difference between our method and that of Bal- primer in the targeted gene and one primer on the P-element linger and Benzer (1) is that our approach obviates the need terminal repeat produces a fragment only ifthe P element had for doing the crosses to mobilize P elements in a laboratory inserted near the gene (Fig. 1). Ifa band is seen, it implies that stock. Instead, we make use of the fact that transposable one of the 200 haploid genomes within the DNA sample has elements are found in natural populations nearly uniformly the appropriate insert. It is then a matter of sequentially distributed throughout the genome (4). Even though there subdividing the descendants of the original 100 males that may be 30-50 copies of the P element per genome of wild-caught flies, the site occupancy is low, meaning that The publication costs ofthis article were defrayed in part by page charge multiple recovery ofthe same insertion is unlikely. Naturally payment. This article must therefore be hereby marked "advertisement" occurring P elements represent a diverse collection of de- in accordance with 18 U.S.C. §1734 solely to indicate this fact. fective or partially deleted elements whose insertional prop- 719 Downloaded by guest on September 29, 2021 720 Genetics: Clark et al. Proc. Natl. Acad. Sci. USA 91 (1994) erties may vary, but defective elements gener.ally retain the looking for P elements near any of several genes, the chance terminal inverted repeats, so they can still be iused to prime that at least one is tagged is very high withjust afew thousand the PCRs and identify tagged sites. Because mo)st incomplete genomes surveyed. Given the approach ofpooling DNA from elements have intact terminal repeats, they can be mobilized 100 males (200 haploid genomes) per initial PCR, excellent to generate imprecise excisions once the insertion is identi- prospects for success should be secured with only 100 fled. There has been a suggestion that A2-3-itnduced P-ele- primary PCRs. ment transposition produces different arrays of insertions depending on the initial line used, and this clustering of MATERIALS AND METHODS insertions does not appear to be present amione naturallv occurring insertions (4). Hence, nature has alrevady done the Fly Collection and Genetic Crosses. During the period from P-element mobilization, and all that is needed is to collect the mid-July to mid-August 1992, Drosophila were collected in flies, allow them to reproduce, and test them:for inserts. about 50 plastic buckets partly filled with mashed rotting There is considerable gain in efficiency that arrises when the peaches and left under the peach trees in the Wolfskill PCRs are multiplexed with multiple primers in erach reaction, orchard of the University of California near Winters, Cali- and we devised a scheme for identifying sets of primers to fornia. Flies were collected in the early morning hours and minimize false priming. False priming is readily detected by again just before sunset. In the laboratory, male D. melano- repeating PCRs with individual target gene primers and by gaster were separated from the females and from Drosophila Southern blot analysis. As in Ballinger and Bi,enzer (1), we simulans (which were about 50% the sample at this time of apply the PCR to genomic DNA isolated from I.ooled groups year) and were placed in groups of 20 into half-pint bottles of 100 males that had already reproduced. 01igonucleotide with 20-30 virgin T(2;3) CyO; TM2/+; ry, Sb females. Note primers complementary to one strand of severad target genes that the F1 progeny will have P elements mobilized (6), but were included with an oligonucleotide primer complementary the rate of excision was low enough that this did not appear to the terminal repeats of the P element. If a i element had to be a problem. The virgin females were between 1 and 5 inserted within about 2 kb of the target gene:, the pair of days old and mated readily with the wild-caught males. It is primers produced a PCR product that was ideantified on an important that there be little opportunity to express differ- agarose gel. We get around the serious prolblem of false ential mating success by the males, because we want an even positives by performing a Southern blot analysis of the representation of all 20 males in the progeny within a bottle.

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