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Mead Lab: Screens

Transposon Hopping

• Sleeping Beauty
• Tol2

Transposon Gene and Enhancer Traps

Transposon Hopping

My laboratory has focused on the use of the Class II DNA 'cut-and-paste' transposons to modify the Xenopus genome. Transposons efficiently integrate into the frog genome and are passed through the germline at the expected Mendelian frequency. The transgenes integrate into the genome without plasmid vector sequences that may cause epigenetic silencing. The primary reason for choosing transposons for manipulating the frog genome is that a transposon integrated in the genome is a substrate for excision and re-integration (remobilization) following re-expression of the cognate transposase enzyme.

Transposon founder animals are generated by co-injecting fertilized embryos at the one-cell stage with a plasmid harboring a transposon transgene and mRNA encoding the transposase. Our efforts have focused on two systems: Sleeping Beauty (SB) and Tol2 - although we are experimenting with a variety of different transposon systems. Using the co-injection strategy to generate founder lines transgenesis rates of approximately 30% are achieved with both SB and Tol2 (see Methods section).

Remobilization of stably integrated transposon transgenes is being pursued by two methods. First, fertilized embryos harvested from transposons transgenic parents are injected with mRNA encoding the cognate transposase enzyme at the one-cell stage. Second, the transposase enzyme can be expressed as a transgene and double transgenic animals, harboring both the transposase enzyme transgene and transposon substrate, are outcrossed to wild type animals and novel reintegration events are scored in the progeny (see figure). The obvious advantage of the second strategy is the simplicity and efficiency. The breeding strategy removes the time-consuming injection process and vastly increases the potential recovery of novel insertion sites. Each outcross can generate thousands of embryos (our record to date is 4,798 swimming tadpoles from a single outcross). Due to the fact that males can be outcrossed more frequently than females (we outcross males every two weeks and females every 8-10 weeks), the majority of our remobilization strategies make use of double transgenic males that are outcrossed to wild type females. With a small cadre of double transgenic males (~12), many thousands of progeny can be generated each week.

Double transgenic strategy for transposon remobilization in the frog. A transgenic frog with a transposon substrate harboring a GFP reporter (green frog) is crossed with a transposase enzyme-expressing frog. The transposase enzyme transgene has a linked fluorescent protein reporter so that the genotype of the animal can be determined by monitoring the eye color. In the example shown above, a gamma cyrstallin promoter-RFP (ΥcRFP) mini-gene was cloned upstream of the transposase enzyme transgene so that transgenic animals could be identified based on RFP expression in the lens. The GFP-positive F3 progeny are examined for remobilization events.

Sleeping Beauty

A member of the Tc1/mariner family of transposable elements, Sleeping Beauty was molecularly reconstructed from an ancient inactive element found in Atlantic Salmon (Ivics et al., (1997) Cell 91(4):501-10). The SB transposase integrates at TA dinucleotides and catalyzes essentially random integration events in the genome. We have shown that the SB system works efficiently in the frog (Xenopus laevis and Xenopus tropicalis); however, the integration events mediated by the co-injection approach are by a non-canonical mechanism that results in the insertion of small-order concatamers at discrete loci in the genome. The concatamers frequently contain parts of the plasmid vector sequence making cloning of these integration sites somewhat challenging. Despite these caveats, SB is an efficient tool for transgenesis in the frog.

Donald A. Yergeau, Michelle R. Johnson Hamlet, Emin Kuliyev, Haiqing Zhu, Joanne R. Doherty, Taylor D. Archer, Andrea P. Subhawong, Marc B. Valentine, Clair M. Kelley and Paul E. Mead. (2009) Transgenesis in Xenopus Using the Sleeping Beauty Transposon System. Developmental Dynamics 238(7):1727-1743.

Joanne R. Doherty, Michelle R. Johnson Hamlet, Emin Kuliyev and Paul E. Mead. (2007) A flk-1 promoter/enhancer reporter transgenic Xenopus laevis generated using the Sleeping Beauty transposon system: an in vivo model for vascular studies. Developmental Dynamics 236(10):2808-2817.

Tol2

Tol2 is a member of the hAT family of transposases and was the first active DNA 'cut-and-paste' transposon system identified in vertebrates. Tol2 was discovered in a population of albino Medaka fish due to integration of the transposon into the tyrosinase gene that resulted in the pigment-less phenotype (Koga et al., (1996) Nature 383(6595):30). We have shown that Tol2 efficiently integrates DNA into the frog genome using the co-injection strategy and that, unlike SB , the insertions are mediated via canonical transposition. Once integrated into the Xenopus tropicalis genome, the Tol2 transposons are effective substrates for remobilization following expression of the transposoase by micro-injecting transgenic one-cell embryos with synthetic mRNA.

Michelle R. Johnson Hamlet, Donald A. Yergeau, Masatoshi Takeda, Masanori Taira, Koichi Kawakami and Paul E. Mead. (2006) Tol2 transposon-mediated transgenesis in Xenopus tropicalis . Genesis 44(9):438-445.

Donald A. Yergeau, Clair M. Kelley, Emin Kuliyev, Haiqing Zhu, Amy K. Sater, Dan E. Wells and Paul E. Mead. (2010) Remobilization of Tol2 transposons in the germline of Xenopus tropicalis . BMC Developmental Biology 10:11. PMCID: PMC2848417.

Transposon Gene and Enhancer Traps

We are using transposons that function as enhancer traps (ET; minimal promoter constructs driving expression of GFP), gene traps (GT; promoter-less GFP reporter with an upstream 5' splice acceptor site) and polyadenylation traps (PAT; minimal promoter driving expression of a fluorescent reporter that lacks a function 3'-UTR region but has a splice donor site cloned downstream of the reporter open reading frame).

Examples of SB-mediated enhancer traps

Sleeping Beauty enhancer trap Xenopus. Embryos were injected at the one-cell stage with transposon pT2βGFP and SB10 mRNA. (i) Ubiquitous expression of GFP in a feeding tadpole. (ii) Half-transgenic frog, presumably due to integration of the transposon in one blastomere at the two-cell stage (insert depicts region of the adult frog that is shown). (iii) GFP expression in the thymus. The eye is in the bottom left corner. (iv) GFP expression in the bone. Three digits of the right forelimb of frog 'G-bone' is shown.

Examples of Tol2 enhancer trap animals are shown in the Transgenic Frogs section.

Last Updated 5/11/10