Proc. Nati. Acad. Sci. USA Vol. 87, pp. 1686-1690, March 1990 Genetics "Site-selected" transposon mutagenesis of Drosophila (P element/polymerase chain reaction/sib selection/singed/transposon mutagenesis) KIM KAISERt AND STEPHEN F. GOODWIN Department of Genetics, University of Glasgow, Church Street, Glasgow G11 5JS, Scotland Communicated by Seymour Benzer, December 4, 1989 (receivedfor review September 11, 1989) ABSTRACT Despite the wide range of techniques that can desired characteristics merely by identifying at each stage the be brought to bear on the study of basic processes in Dro- sub-population in which at least one individual is laying eggs sophila, there are still deficiencies in our armory. One of these with the appropriate chromosomal configuration. is an ability to select mutants in cases where the gene is known and has been cloned, but where we are ignorant of the associated phenotype. We describe here a solution to this MATERIALS AND METHODS problem as applied to a model system, the singed (sn) locus. Flies and DNAs. CFL3 and CFL5 (sn strains, ref. 4), Our method is a combination of classical genetics and molec- Birm-2; ry5 and w; SbP[ry'A2,3]/TM6 (5), ir2 and Harwich ular biology: sib selection plus the polymerase chain reaction. (strong P strains; ref. 6) and P-cytotype balancer strains were We have used the method to isolate rare individuals with obtained from J. Paterson and K. O'Hare (Imperial College P-element-induced alleles of sn merely by recognition of the of Science, Technology and Medicine, London), as was the DNA structures induced at the locus by transposon insertion. sn9 DNA clone. Consult ref. 7 for a description of the genetic Phenotypic criteria were used only retrospectively to verify our markers and fly strains not described here. diagnoses. There are obvious implications of this technique for Mutagenesis. Both strategies began with a cross involving the mutagenesis of other organisms. 20 males and 50 females. Batches of 40 F1 males were mated with 100 females and transferred to new bottles regularly to There is a rapidly growing number of loci in Drosophila for avoid overcrowding. This generated more than sufficient F2 which cloned genes are available but for which we neither females for our experiments. have phenotypic information nor can be sure what kind of Strategy A. F1 male progeny of ir2 males and Oregon R screening procedure would be likely to provide it. Many (M-cytotype) females were mated with P-cytotype FM7/ Drosophila genes, for example, have been cloned by homol- Basc females. Batches of100-200 F2 virgin females (sn?/FM7 ogy with those of other organisms (1); other genes have been or sn?/Basc) were placed in small cages with 50 young cloned on the basis that they exhibit an interesting pattern of Oregon R males, matured for at least 24 hr, and thereafter differential gene expression when two tissues or develop- eggs were collected for 24 hr on yeasted grape juice agarose mental stages are compared (e.g., ref. 2). We can sequence plates (all at 200C). such genes, study their patterns of expression in detail, and Strategy B. Birm-2; ry5-' males, homozygous for a second generally adduce a wealth of circumstantial evidence in chromosome bearing 17 defective P elements, were mated at support of a belief that they play an interesting role in the 16'C with w; Sb P[ry+A2,3]/TM6 females (5). P[ry'A2,3] biology of the fly. Without a phenotype, however, we are provides a nonmobilizable source of transposase. F1 male severely handicapped in the scope of our investigations, progeny (w/Y; Birm-2/+; Sb P[ry+A2,3]/ry5os) were mated being unable either to demonstrate function by a study of with Oregon R females at 18'C. Approximately 900 Sb+ F2 individuals in which gene expression has been perturbed or virgin females were placed in a cage at 20'C with 200 Oregon to make use of the most important characteristic of the fly, R males and matured, and their eggs were collected as above. the wealth of accumulated genetic knowledge. We describe DNA Isolation. Eggs were washed with tap water and here a simple, fast, and general solution to this problem as homogenized in 500 ttl or more of 10 mM Tris HCl, pH 7.4/10 applied to a model system, the singed (sn) locus. mM EDTA/60 mM NaCl/0.15 mM spermine/0.15 mM sper- The logic of our method is as follows: The polymerase midine/0.5% Triton X-100. The homogenate was extracted chain reaction (PCR) allows the detection, even within small once with phenol/chloroform [1:1 (vol/vol)], ethanol- amounts of DNA, of specific DNA sequences and configu- precipitated, and taken up in an aqueous solution of RNase rations of sequences (3). Thus, should a transposon lie A (10 ,ug/ml). After a 30-min incubation at 37°C, two further within, or close to, any genetic locus we can detect this phenol/chloroform extractions, one chloroform extraction, chromosomal state by PCR between one primer specific for and a further precipitation, the DNA was taken up in 100- the transposon and one primer specific for the genetic locus. 1000 ,u of 10 mM Tris'HCl, pH 7.8/1 mM EDTA. Approx- Moreover, due to the extreme sensitivity of the PCR, we can imately 1 ,ug of egg DNA was obtained per F2 female. Adult detect one individual with such a chromosomal configuration DNA was prepared essentially by the same method. within a large excess of flies that do not carry a transposon PCR and Analysis of Amplified DNA. HPLC-purified oli- at the locus. We have exploited these findings to detect and gonucleotide primers were provided by the Oswell DNA isolate rare individuals (within a population of mutagenized Service (Edinburgh University). Their approximate locations flies) that carry a transposable P element inserted into a target are shown in Fig. 1. [PI is 5'-CGACGGGACCACCTTAT- gene, in this case sn. Rather than sacrifice the flies them- GTTATTTCATCATG, derived from the 31-base-pair (bp) selves we have carried out PCR on eggs laid by mutagenized terminal inverted repeat sequence of the P element (8). [snA] flies. Then, by subdivision of the population in successive is 5'-GTCTGTCGTCACACCCTTCACTTCGCC, derived stages, we have been able to "home-in" on a rare fly with the from the sequence beginning 150 bp upstream of the right- hand EcoRI site of Fig. 1 (within the sn gene). [snB] is The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: PCR, polymerase chain reaction. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 1686 Downloaded by guest on October 1, 2021 Genetics: Kaiser and Goodwin Proc. Nati. Acad. Sci. USA 87 (1990) 1687 * PCR primer [PI P-element [PI which the precise location ofthe relevant P element is known (ref. 4; J. Paterson and K. O'Hare, personal communication). "hot-spot" Fig. 2 shows that amplification products ofthe expected sizes lkb and hybridization characteristics are produced when mutant DNA is amplified with [P] and [snA] primers. "Spurious" [snB] snA] . __ amplification also occurs in both sn and sn' DNAs. Further r I evidence of spurious products is shown in Fig. 3. They are EcoRI Hindi I11 Xbal EcoRI generated in all strains tested, even when the primers are used singly (but not in their absence or in the absence of DNA; data not shown). Moreover, their generation is inde- pendent of the number of P elements in the genome (Fig. 3) and ofthe PCR annealing temperature (in the range 50-70'C; FIG. 1. Promoter-proximal and 5' flanking region of the sn locus data not shown). We are and in the Oregon R and w; SbP[A2,3]/TM6 stocks used in this study (4). ignorant of their origin, their Several independent sites of P-element insertion have been docu- intensity varies from experiment to experiment. Since they mented within the hot-spot region. sn9 is a cloned EcoRI fragment. are not detected by hybridization and do not confuse our PCR primers are shown in square brackets. analysis, we do not consider them further. Can a Rare sn Fly Be Detected Within a Population Of sn' 5'-GCTGCCCTTCTAATCCTCGCGTCC, derived from the Flies? Serial dilution of CFL5 DNA into Oregon R DNA, and sequence beginning 11 bp downstream of the HindIII site of amplification as above, shows that the equivalent of 1 fly in Fig. 1 (5' to the sn gene). Egg DNA (-0.2 ,ug per 10 F2 females 1000 can be detected easily by hybridization, if with some or 2 pug per 100-900 F2 females) was amplified in 20 A.l of 50 difficulty in the stained gel (Fig. 4). Emboldened by this mM KCI/10 mM Tris-HCl, pH 8.3/1.5 mM MgCl2/0.1% result, we proceeded to generate mutant flies by strategy A gelatin/200 ,uM dATP/200 puM dCTP/200 ,M dGTP/200 AM and to screen for F2 females that were laying eggs with a P dTTP/the two primers, each at 1 ,uM/1 unit of Thermus element insertion at the sn locus. Approximately 4500 fe- aquaticus DNA polymerase (Cambio, U.K.). The samples, males were screened in batches of 100-200, from which we overlain with 20 ,.l of mineral oil (Merck) in 1.5-ml micro- detected and isolated three sn lines (snGl-3; see below). centrifuge tubes, were placed in an Intelligent Heating Block However, although we had naively assumed that the male (Cambio); heated to 940C for 3 min; taken through 30 cycles progeny of a selected F2 female would be of only two types, of 1 min at 500C, 3 min at 700C, and 1 min at 940C; and finally those with a balancer X chromosome and those with an sn incubated for 5 min at 50'C and for 20 min at 700C.