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Genetics. Aug 2004; 167(4): 1801–1811.
PMCID: PMC1470976

A piggyBac transposon gene trap for the analysis of gene expression and function in Drosophila.

Abstract

P-element-based gene and enhancer trap strategies have provided a wealth of information on the expression and function of genes in Drosophila melanogaster. Here we present a new vector that utilizes the simple insertion requirements of the piggyBac transposon, coupled to a splice acceptor (SA) site fused to the sequence encoding enhanced green fluorescent protein (EGFP) and a transcriptional terminator. Mobilization of the piggyBac splice site gene trap vector (PBss) was accomplished by heat-shock-induced expression of piggyBac transposase (PBase). We show that insertion of PBss into genes leads to fusions between the gene's mRNA and the PBss-encoded EGFP transcripts. As heterozygotes, these fusions report the normal pattern of expression of the trapped gene. As homozygotes, these fusions can inactivate the gene and lead to lethality. Molecular characterization of PBss insertion events shows that they are single copy, that they always occur at TTAA sequences, and that splicing utilizes the engineered splice site in PBss. In those instances where protein-EGFP fusions are predicted to occur, the subcellular localization of the wild-type protein can be inferred from the localization of the EGFP fusion protein. These experiments highlight the utility of the PBss system for expanding the functional genomics tools that are available in Drosophila.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bond BJ, Davidson N. The Drosophila melanogaster actin 5C gene uses two transcription initiation sites and three polyadenylation sites to express multiple mRNA species. Mol Cell Biol. 1986 Jun;6(6):2080–2088. [PMC free article] [PubMed]
  • Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993 Jun;118(2):401–415. [PubMed]
  • Cary LC, Goebel M, Corsaro BG, Wang HG, Rosen E, Fraser MJ. Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology. 1989 Sep;172(1):156–169. [PubMed]
  • Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802–805. [PubMed]
  • Chung YT, Keller EB. Regulatory elements mediating transcription from the Drosophila melanogaster actin 5C proximal promoter. Mol Cell Biol. 1990 Jan;10(1):206–216. [PMC free article] [PubMed]
  • Elick TA, Bauser CA, Principe NM, Fraser MJ., Jr PCR analysis of insertion site specificity, transcription, and structural uniformity of the Lepidopteran transposable element IFP2 in the TN-368 cell genome. Genetica. 1996 Mar;97(2):127–139. [PubMed]
  • Furlong EE, Profitt D, Scott MP. Automated sorting of live transgenic embryos. Nat Biotechnol. 2001 Feb;19(2):153–156. [PubMed]
  • Gossler A, Joyner AL, Rossant J, Skarnes WC. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science. 1989 Apr 28;244(4903):463–465. [PubMed]
  • Handler AM, Harrell RA., 2nd Germline transformation of Drosophila melanogaster with the piggyBac transposon vector. Insect Mol Biol. 1999 Nov;8(4):449–457. [PubMed]
  • Jackson Stephen M, Berg Celeste A. An A-kinase anchoring protein is required for protein kinase A regulatory subunit localization and morphology of actin structures during oogenesis in Drosophila. Development. 2002 Oct;129(19):4423–4433. [PubMed]
  • Leighton PA, Mitchell KJ, Goodrich LV, Lu X, Pinson K, Scherz P, Skarnes WC, Tessier-Lavigne M. Defining brain wiring patterns and mechanisms through gene trapping in mice. Nature. 2001 Mar 8;410(6825):174–179. [PubMed]
  • Li X, Heinrich JC, Scott MJ. piggyBac-mediated transposition in Drosophila melanogaster: an evaluation of the use of constitutive promoters to control transposase gene expression. Insect Mol Biol. 2001 Oct;10(5):447–455. [PubMed]
  • Li Z, Rossi EA, Hoheisel JD, Kalderon D, Rubin CS. Generation of a novel A kinase anchor protein and a myristoylated alanine-rich C kinase substrate-like analog from a single gene. J Biol Chem. 1999 Sep 17;274(38):27191–27200. [PubMed]
  • Lobo N, Li X, Fraser MJ., Jr Transposition of the piggyBac element in embryos of Drosophila melanogaster, Aedes aegypti and Trichoplusia ni. Mol Gen Genet. 1999 Jun;261(4-5):803–810. [PubMed]
  • Long M, Deutsch M. Association of intron phases with conservation at splice site sequences and evolution of spliceosomal introns. Mol Biol Evol. 1999 Nov;16(11):1528–1534. [PubMed]
  • Long Manyuan, Deutsch Michael, Wang Wen, Betrán Esther, Brunet Frédéric G, Zhang Jianming. Origin of new genes: evidence from experimental and computational analyses. Genetica. 2003 Jul;118(2-3):171–182. [PubMed]
  • Lukacsovich T, Asztalos Z, Awano W, Baba K, Kondo S, Niwa S, Yamamoto D. Dual-tagging gene trap of novel genes in Drosophila melanogaster. Genetics. 2001 Feb;157(2):727–742. [PMC free article] [PubMed]
  • Morin X, Daneman R, Zavortink M, Chia W. A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):15050–15055. [PMC free article] [PubMed]
  • Mount SM, Burks C, Hertz G, Stormo GD, White O, Fields C. Splicing signals in Drosophila: intron size, information content, and consensus sequences. Nucleic Acids Res. 1992 Aug 25;20(16):4255–4262. [PMC free article] [PubMed]
  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. [PubMed]
  • Niwa H, Araki K, Kimura S, Taniguchi S, Wakasugi S, Yamamura K. An efficient gene-trap method using poly A trap vectors and characterization of gene-trap events. J Biochem. 1993 Mar;113(3):343–349. [PubMed]
  • Bellen HJ, O'Kane CJ, Wilson C, Grossniklaus U, Pearson RK, Gehring WJ. P-element-mediated enhancer detection: a versatile method to study development in Drosophila. Genes Dev. 1989 Sep;3(9):1288–1300. [PubMed]
  • O'Brochta DA, Atkinson PW. Transposable elements and gene transformation in non-drosophilid insects. Insect Biochem Mol Biol. 1996 Sep-Oct;26(8-9):739–753. [PubMed]
  • Bier E, Vaessin H, Shepherd S, Lee K, McCall K, Barbel S, Ackerman L, Carretto R, Uemura T, Grell E, et al. Searching for pattern and mutation in the Drosophila genome with a P-lacZ vector. Genes Dev. 1989 Sep;3(9):1273–1287. [PubMed]
  • O'Kane CJ, Gehring WJ. Detection in situ of genomic regulatory elements in Drosophila. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9123–9127. [PMC free article] [PubMed]
  • Blackman RK, Koehler MM, Grimaila R, Gelbart WM. Identification of a fully-functional hobo transposable element and its use for germ-line transformation of Drosophila. EMBO J. 1989 Jan;8(1):211–217. [PMC free article] [PubMed]
  • Rossi EA, Li Z, Feng H, Rubin CS. Characterization of the targeting, binding, and phosphorylation site domains of an A kinase anchor protein and a myristoylated alanine-rich C kinase substrate-like analog that are encoded by a single gene. J Biol Chem. 1999 Sep 17;274(38):27201–27210. [PubMed]
  • Wilson C, Pearson RK, Bellen HJ, O'Kane CJ, Grossniklaus U, Gehring WJ. P-element-mediated enhancer detection: an efficient method for isolating and characterizing developmentally regulated genes in Drosophila. Genes Dev. 1989 Sep;3(9):1301–1313. [PubMed]
  • Skarnes WC, Auerbach BA, Joyner AL. A gene trap approach in mouse embryonic stem cells: the lacZ reported is activated by splicing, reflects endogenous gene expression, and is mutagenic in mice. Genes Dev. 1992 Jun;6(6):903–918. [PubMed]
  • Zambrowicz BP, Friedrich GA, Buxton EC, Lilleberg SL, Person C, Sands AT. Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells. Nature. 1998 Apr 9;392(6676):608–611. [PubMed]
  • Skarnes WC, Moss JE, Hurtley SM, Beddington RS. Capturing genes encoding membrane and secreted proteins important for mouse development. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6592–6596. [PMC free article] [PubMed]

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