Abstract
The class II DNA “cut-and-paste” transposons have been used to efficiently modify the Xenopus genome for transgenesis applications. Once integrated, the transposon is an effective substrate for excision and re-integration (remobilization) elsewhere in the genome by simply supplying the transposase enzyme in trans. We have used two methods to remobilize transposons resident in the frog genome: micro-injection of transposase mRNA at the one-cell stage and expression of the enzyme in the germline from a transgene. Double-transgenic frogs (hoppers) that harbor transgenes for both the substrate transposon and the transposase enzyme are outcrossed to wild-type animals and the progeny are scored for changes in reporter gene expression. Although both methods work effectively to remobilize transposons, the breeding-mediated strategy eliminates the time-consuming micro-injection step; novel integration events are produced by simply outcrossing the hopper frogs. As each outcross of Xenopus tropicalis typically produces 2,000, or more, progeny, this method can be used to perform large-scale insertional mutagenesis screens in this highly tractable developmental model system.
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References
Clark KJ, Geurts AM, Bell JB, Hackett PB (2004) Transposon vectors for gene-trap insertional mutagenesis in vertebrates. Genesis 39:225–233
Ding S, Wu X, Li G, Han M, Zhuang Y, Xu T (2005) Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell 122:473–483
Dupuy AJ, Clark K, Carlson CM, Fritz S, Davidson AE, Markley KM, Finley K, Fletcher CF, Ekker SC, Hackett PB, Horn S, Largaespada DA (2002) Mammalian germ-line transgenesis by transposition. Proc Natl Acad Sci U S A 99:4495–4499
Grabher C, Henrich T, Sasado T, Arenz A, Wittbrodt J, Furutani-Seiki M (2003) Transposon-mediated enhancer trapping in medaka. Gene 322:57–66
Horn C, Wimmer EA (2000) A versatile vector set for animal transgenesis. Dev Genes Evol 210:630–637
Ivics Z, Izsvak Z (2004) Transposable elements for transgenesis and insertional mutagenesis in vertebrates: a contemporary review of experimental strategies. Methods Mol Biol 260:255–276
Izsvak Z, Ivics Z, Plasterk RH (2000) Sleeping Beauty, a wide host-range transposon vector for genetic transformation in vertebrates. J Mol Biol 302:93–102
Johnson Hamlet MR, Mead PE (2003) Sleeping Beauty and Xenopus: transposons as genetic tools. Curr Genomics 4:687–697
Kawakami K (2007) Tol2: a versatile gene transfer vector in vertebrates. Genome Biol 8(suppl 1):S7
Koga A, Cheah FS, Hamaguchi S, Yeo GH, Chong SS (2008) Germline transgenesis of zebrafish using the medaka Tol1 transposon system. Dev Dyn 237:2466–2474
Korzh V (2007) Transposons as tools for enhancer trap screens in vertebrates. Genome Biol 8(suppl 1):S8
Miskey C, Izsvak Z, Kawakami K, Ivics Z (2005) DNA transposons in vertebrate functional genomics. Cell Mol Life Sci 62:629–641
Takeda J, Keng VW, Horie K (2007) Germline mutagenesis mediated by Sleeping Beauty transposon system in mice. Genome Biol 8(suppl 1):S14
Yergeau DA, Mead PE (2007) Manipulating the Xenopus genome with transposable elements. Genome Biol 8(suppl 1):S11
Collier LS, Largaespada DA (2007) Transposons for cancer gene discovery: Sleeping Beauty and beyond. Genome Biol 8(suppl 1):S15
Dupuy AJ, Fritz S, Largaespada DA (2001) Transposition and gene disruption in the male germline of the mouse. Genesis 30:82–88
Largaespada DA (2009) Transposon mutagenesis in mice. Methods Mol Biol 530:379–390
Su Q, Prosser HM, Campos LS, Ortiz M, Nakamura T, Warren M, Dupuy AJ, Jenkins NA, Copeland NG, Bradley A, Liu P (2008) A DNA transposon-based approach to validate oncogenic mutations in the mouse. Proc Natl Acad Sci U S A 105:19904–19909
Keng VW, Ryan BJ, Wangensteen KJ, Baciunas D, Schmedt C, Ekker SC, Largaespada DA (2009) Efficient transposition of Tol2 in the mouse germline. Genetics 183:1565–1573
Wang W, Bradley A, Huang Y (2009) A piggyBac transposon-based genome-wide library of insertionally mutated Blm-deficient murine ES cells. Genome Res 19:667–673
Kitada K, Keng VW, Takeda J, Horie K (2009) Generating mutant rats using the Sleeping Beauty transposon system. Methods 49(3):236–242
Lu B, Geurts AM, Poirier C, Petit DC, Harrison W, Overbeek PA, Bishop CE (2007) Generation of rat mutants using a coat color-tagged Sleeping Beauty transposon system. Mamm Genome 18:338–346
Urasaki A, Asakawa K, Kawakami K (2008) Efficient transposition of the Tol2 transposable element from a single-copy donor in zebrafish. Proc Natl Acad Sci U S A 105:19827–19832
Choo BG, Kondrichin I, Parinov S, Emelyanov A, Go W, Toh WC, Korzh V (2006) Zebrafish transgenic enhancer TRAP line database (ZETRAP). BMC Dev Biol 6:5
Kondrychyn I, Garcia-Lecea M, Emelyanov A, Parinov S, Korzh V (2009) Genome-wide analysis of Tol2 transposon reintegration in zebrafish. BMC Genomics 10:418
Yergeau DA, Kelley CM, Kuliyev E, Zhu H, Sater AK, Wells DE, Mead PE (2010) Remobilization of Tol2 transposons in Xenopus tropicalis. BMC Dev Biol 10:11
Bestor TH (2005) Transposons reanimated in mice. Cell 122:322–325
Collier LS, Carlson CM, Ravimohan S, Dupuy AJ, Largaespada DA (2005) Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Nature 436:272–276
Dupuy AJ, Akagi K, Largaespada DA, Copeland NG, Jenkins NA (2005) Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature 436:221–226
Rad R, Rad L, Wang W, Cadinanos J, Vassiliou G, Rice S, Campos LS, Yusa K, Banerjee R, Li MA, de la Rosa J, Strong A, Lu D, Ellis P, Conte N, Yang FT, Liu P, Bradley A (2010) PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice. Science 330:1104–1107
Vassiliou G, Rad R, Bradley A (2010) The use of DNA transposons for cancer gene discovery in mice. Methods Enzymol 477:91–106
Parinov S, Kondrichin I, Korzh V, Emelyanov A (2004) Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo. Dev Dyn 231:449–459
Hirsch N, Zimmerman LB, Grainger RM (2002) Xenopus, the next generation: X. tropicalis genetics and genomics. Dev Dyn 225:422–433
Balciunas D, Davidson AE, Sivasubbu S, Hermanson SB, Welle Z, Ekker SC (2004) Enhancer trapping in zebrafish using the Sleeping Beauty transposon. BMC Genomics 5:62
Balciunas D, Ekker SC (2005) Trapping fish genes with transposons. Zebrafish 1:335–341
Bronchain OJ, Hartley KO, Amaya E (1999) A gene trap approach in Xenopus. Curr Biol 9:1195–1198
Kawakami K (2005) Transposon tools and methods in zebrafish. Dev Dyn 234:244–254
Kotani T, Nagayoshi S, Urasaki A, Kawakami K (2006) Transposon-mediated gene trapping in zebrafish. Methods 39:199–206
Sivasubbu S, Balciunas D, Amsterdam A, Ekker SC (2007) Insertional mutagenesis strategies in zebrafish. Genome Biol 8(suppl 1):S9
Urasaki A, Kawakami K (2009) Analysis of genes and genome by the tol2-mediated gene and enhancer trap methods. Methods Mol Biol 546:85–102
Salminen M, Meyer BI, Gruss P (1998) Efficient poly A trap approach allows the capture of genes specifically active in differentiated embryonic stem cells and in mouse embryos. Dev Dyn 212:326–333
Clark KJ, Balciunas D, Pogoda HM, Ding Y, Westcot SE, Bedell VM, Greenwood TM, Urban MD, Skuster KJ, Petzold AM, Ni J, Nielsen AL, Patowary A, Scaria V, Sivasubbu S, Xu X, Hammerschmidt M, Ekker SC (2011) In vivo protein trapping produces a functional expression codex of the vertebrate proteome. Nat Methods 8:506–515
Craig NL, Craigie R, Gellert M, Lambowitz AM (2002) Mobile DNA II. ASM Press, Washington, DC
Liu G, Geurts AM, Yae K, Srinivasan AR, Fahrenkrug SC, Largaespada DA, Takeda J, Horie K, Olson WK, Hackett PB (2005) Target-site preferences of Sleeping Beauty transposons. J Mol Biol 346:161–173
Bauser CA, Elick TA, Fraser MJ (1999) Proteins from nuclear extracts of two lepidopteran cell lines recognize the ends of TTAA-specific transposons piggyBac and tagalong. Insect Mol Biol 8:223–230
Yergeau DA, Kuliyev E, Mead PE (2007) Injection-mediated transposon transgenesis in Xenopus tropicalis and the identification of integration sites by modified extension primer tag selection (EPTS) linker-mediated PCR. Nat Protoc 2:2975–2986
Waldner C, Roose M, Ryffel GU (2007) Marking transgenic Xenopus froglets with passive micro transponders. Transgenic Res 16:539–540
Yergeau DA, Kelley CM, Zhu H, Kuliyev E, Mead PE (2010) Transposon transgenesis in Xenopus. Methods 51:92–100
Hellsten U, Harland RM, Gilchrist MJ, Hendrix D, Jurka J, Kapitonov V, Ovcharenko I, Putnam NH, Shu S, Taher L, Blitz IL, Blumberg B, Dichmann DS, Dubchak I, Amaya E, Detter JC, Fletcher R, Gerhard DS, Goodstein D, Graves T, Grigoriev IV, Grimwood J, Kawashima T, Lindquist E, Lucas SM, Mead PE, Mitros T, Ogino H, Ohta Y, Poliakov AV, Pollet N, Robert J, Salamov A, Sater AK, Schmutz J, Terry A, Vize PD, Warren WC, Wells D, Wills A, Wilson RK, Zimmerman LB, Zorn AM, Grainger R, Grammer T, Khokha MK, Richardson PM, Rokhsar DS (2010) The genome of the Western clawed frog Xenopus tropicalis. Science 328:633–636
Acknowledgements
This work was supported by NIH grants HD042994 and MH079381 and by the American Lebanese and Syrian Associated Charities (ALSAC) to PEM.
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Yergeau, D.A., Kelley, C.M., Zhu, H., Kuliyev, E., Mead, P.E. (2012). Forward Genetic Screens in Xenopus Using Transposon-Mediated Insertional Mutagenesis. In: HOPPLER, S., Vize, P. (eds) Xenopus Protocols. Methods in Molecular Biology, vol 917. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-992-1_6
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DOI: https://doi.org/10.1007/978-1-61779-992-1_6
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