Copyight 0 1996 by the Genetics Society of America Germline Transformation of Drosophila Virilis Mediated by the Transposable Element hobo Elena R. Lozovskaya,* Dmitry I. Nurminsky,* Daniel L. Had* and David T. Sullivan’ *Department of Organismic and Euolutionaly Biology, Haruard University, Cambridge, Massachusetts 02138 and tDepartment of Biology, Syracuse University, Syracuse, New York 13244-0001 Manuscript received July 17, 1995 Accepted for publication September 21, 1995 ABSTRACT A laboratory strain of Drosophila uirilis was genetically transformed with a hobo vector carrying the miniwhite cassette using a helper plasmid with an hsp7Odriven hobo transposasecoding sequence. The rate of transformation was 0.5% per fertile GO animal. Three transgenic insertions were cloned and characterized and found to be authentic hobo insertions. These results, together with the known wide- spread distribution of hobo in diverse insect species, suggest that hobo and related transposable elements may be of considerable utility in the germline transformation of insects other than D. melanogaster. HERE is at present considerable interest in the Thusfar, most transformation experiments have T application of germline transformation to the ge- been carried out with D. melanogaster as a model organ- netic manipulation of arthropod genomes, especially ism. One advantage is that the experimental procedures those of agricultural pests and vectors of human dis- for injection of DNA and the treatment of embryos are ease (KIDWELL 1993; WARRENand CRAMPTON 1994). well established (SPRADLING1986). Another advantage For a number of reasons, transposable elements are is that any one of a number of genetic marker systems strong candidates as potential vectors. An experimen- can be used in vector construction and transformation. tal paradigm already exists in thegermline transforma- A third advantage is that the method is known to work, tion of Drosophila melanogaster (RUBINand SPRADLING which means that the injected DNA must persist long 1982; BLACKMANet al. 1989; LIDHOLMet al. 1993). enough to get into the nucleus and become incorpo- Some types of transposable elements are distributed rated into the genome. widely among diverse insects andother organisms It is not known, however, how representative D. mela- (LIDHOLMet al. 1991; ROBERTSON1993), suggesting nogaster may be of arthropods in general, or even how that whatever genetic functions are required of the representative it may be of other drosophilids. There host genome are either minimalor highly conserved. is considerable variation even among strains of D. mela- In vivo assays based on excision from or transposition nogaster in their tolerance of the experimental proce- between transposon-bearing plasmids affords addi- dures and their ability to be transformed. In species tional evidence for a wide host range for some trans- other than D. melanogaster, the embryos may be less posable elements (O’BROCHTAet al. 1991,1994;ATKIN- tolerant of the experimental manipulations necessary SON et al. 1993; HANDLERand GOMEZ1995). Some for transformation. In some organisms, the injected transposable elements found in distantly related spe- DNA may be degraded by nucleases in the cytoplasm cies give evidence of horizontal transmission, includ- and rendered ineffectual. In others, the injected DNA ing mariner-like elements (MARUYAMA and HARTL1991; may be less likely to get into the nucleus or to become ROBERTSONand MACLEOD1993; LOHEet al. 1994; GAR- incorporated into the chromatin.These are all key vari- CIA-FERNANDEZet al. 1995), P elements (DANIELSet al. ables that, in the absence of direct experimental evi- 1990; KIDWELL 1992; CLARKet al. 1994),and hobo (WAR- dence, can only be objects of speculation. REN et al. 1994, 1995). Finally, transposable elements The potential problems caused by variability among of various typeshave been shown to be capable of species provides a strong incentive to investigate the supporting the incorporation of exogenous DNA into feasibility of germline transformation in a variety of the germline of arthropods other than their species insects other than D. melanogaster. Among drosophi- of origin (BRENNANet al. 1984; SCAVARDAand HARTL lids, a strongcase can be made for theutility of germ- 1984; DANIELSet al. 1985; GARZAet al. 1991; SIMONELIG line transformationin D.virilis. With a divergence time and ANXOLABEHERE1991; LIDHOLMet al. 1993; LOUK- of 40 million years (RUSSOet al. 1995), D. virilzs and EMS et al. 1995; D. A. O’BROCHTA,W. D. WARREN,K. J. D. melanogaster have an evolutionary separation that is SAVILLEand P. W. ATKINSON,unpublished results). greatenough that most nonconservednucleotides have had at least one opportunity to become substi- Corresponding authort Daniel L. Hartl, Department of Organismic and Evolutionary Biology, Harvard University, 16 Divinity Ave., Cam- tuted, yet the separation is not so great that the identi- bridge, MA 02138. E-mail: [email protected] fication of homologous genes is compromised. Fur- Genetics 142: 173-177 (January, 1996) 174 Lozovskaya E. R. et al. thermore, the genetic transformation of genes from Sau3A and ligation with the XhoI arms of the replacement D. uirilis into D. melanogaster is coming into increasing vector LambdaScan after half-filling the cohesive ends with the Klenow fragment of DNA polymerase. Lambdascan is a prominence for studies of gene regulation and func- specialized derivative of Lambdam (Stratagene) designed tional divergence. It would contribute significantly to to cloneinserts in thesize range from 8 to 12 kb (NURMINSKY the power of this approach if the reciprocal transfor- and HARTL 1995). The ligated DNA molecules were pack- mation of genes from D. melanogaster into D. virilis aged in vitro using Gigapack XL extracts (Stratagene). The could also be carried out. resulting libraries were screened with a hobo probe obtained byPCR amplification of nucleotides 290-860 in the hobo In this paper, we report that D. virilis can be geneti- sequence. From the positive lambda clones, plasmids were cally transformed with vectors based on the transpos- obtained by in vivo excision with the ExAssist/SOLR system able element hobo. These vectors have previously been (Stratagene) and sequenced with the DyeDeoxyTerminator demonstrated to support germline transformation of D. reagents on an Applied Biosystems 373A automated DNA melanogaster (BLACKMANet al. 1989). Thegenetic marker sequencer using the hobwpecific primers Sul-5a (5”CAGCA- GGCCTCGGGTGGTTTT-3’), Sul-7 (5’-ACTCGTAACAAAA- usedin the transformation was a miniwhite cassette, ACACAACG3‘), and SUI-8 (5”CCGAGTATTTTTGGAAAC- which we reasoned would be able to complement a ACC-3’). The unique insertion in strain Dv[hawll-12 was white mutation in D. virilis. Three transgenic insertions recovered and the ends sequenced.Two different insertions were examined at themolecular level and foundto have were recovered fromthe multiply transformed strain the sequencecharacteristics expected of a conventional Dv[hawl] -131. hobo insertion. RESULTS MATERIALSAND METHODS Efficiency of transformation:A total of 1804 D. virilis Drosophila strains: The X-linked w mutation in D. virilis w embryos were injected with the hobo miniwhite trans- was obtained from JEAN DAVID.The eye-color phenotype is formation vector Ei[w+, hawl] (Caw et al. 1991) and bleached white. Based on its linkage to the X chromosome the helper elementHSH2. Among these, 477 GO adults and the phenotypeof the mutantflies, the mutationhas been presumed to be homologous to the X-linked w gene in D. were obtained, of which 398 were fertile. Two fertile melanogaster (ALEXANDER 1976). Further evidence for this in- GO parents yielded progeny that were putative trans- ference was obtained by isolation of a D. vidis P1 clone, using formants, for a rate of 0.5% transformants per fertile a probe from the D. melanogaster w gene, and demonstrating GO. Strains derived from the putative transformants that the wbearing PI clone undergoes in situ hybridization were designated Dv[hawl] -12and Dv[hawl] -131.The with the polytene chromosome region containing theD. virilis w gene (LOZOVSKAYAet al. 1993). The transformation experi- eye-color phenotype of Dv[hawl] -12is very weak and ments described here demonstrate the homology directly by appears variegated. The phenotype of Dv[hawl] -131 is complementation. dark red but distinct from wild type. As shown below, Transformation vector: The hobebased transformation vec- Dv[hawl] -12has a single insertion of the hobo-miniwhite tors have been described previously. The helper elementwas construct whereas Dv[hawl] -131has three different in- a plasmid containing the hsp70:hobo fusion HSHP described in Guw and GELBART(1994; their Figure 1, p. 1637). The sertions. transformation vector was H[w+, hawl] described in Gum et Evidencefor hobo-mediated transformation: The al. (1991; their Figure 4B). Theminiwhite insert in this plasmid first line of evidence for the authenticity of the putative is oriented with the transcriptional start near the 3’ end of transformants was the observation that genomic DNA hobo. In D. melanogaster, the miniwhite gene confers an interme- from both Dv[hawl]-l2 and Dv[hawl]-131, butnot diate w+ phenotype (PIRROTTAet al. 1985). Germline transformation: Plasmid DNA was purified twice from untransformed controls, could support PCR am- in cesium chloride density gradients and used for injection plification using oligonucleotide primers to amplify spe- of embryos. D. virilis w embryos were