Remobilization of Tol2 transposons in Xenopus tropicalis

Abstract

BackgroundThe Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring Tol2 element from Oryzias latipes efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, Xenopus tropicalis. Tol2 transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency.ResultsTo test whether Tol2 transposons integrated in the Xenopus tropicalis genome are substrates for remobilization, we injected in vitro transcribed Tol2 mRNA into one-cell embryos harbouring a single copy of a Tol2 transposon. Integration site analysis of injected embryos from two founder lines showed at least one somatic remobilization event per embryo. We also demonstrate that the remobilized transposons are transmitted through the germline and re-integration can result in the generation of novel GFP expression patterns in the developing tadpole. Although the parental line contained a single Tol2 transposon, the resulting remobilized tadpoles frequently inherit multiple copies of the transposon. This is likely to be due to the Tol2 transposase acting in discrete blastomeres of the developing injected embryo during the cell cycle after DNA synthesis but prior to mitosis.ConclusionsIn this study, we demonstrate that single copy Tol2 transposons integrated into the Xenopus tropicalis genome are effective substrates for excision and random re-integration and that the remobilized transposons are transmitted through the germline. This is an important step in the development of 'transposon hopping' strategies for insertional mutagenesis, gene trap and enhancer trap screens in this highly tractable developmental model organism.

Breeding Based Remobilization of Tol2 Transposon in Xenopus tropicalis

“…This “cut and paste” transposon inserts canonically at a random heterogenic sequence, often at multiple loci, and creates a signature eight base pair (bp) target site duplication (TSD) [18]. Tol2 has applications for Xenopus transgenesis [19-21], has demonstrated “local hopping” [22-25], and has been shown to favor insertion into transcriptional regions of genes [24-26], all properties which make it highly attractive as a gene and enhancer trapping tool in frog.…”

“…In Drosophila and Zebrafish, transposon remobilization and screening of transgenic lines is common and effective [23,24,28-30]. In X. tropicalis , remobilization of transposons has been demonstrated using two methods: 1) microinjection of transposase mRNA (Tol2) [22] and 2) breeding transposon lines to transposase lines (Sleeping Beauty) [27]. …”

“…In the microinjection method [22], in vitro transcribed Tol2 mRNA injected in embryos bearing the Tol2 transposon resulted in a very high remobilization efficiency (18/18 injected embryos showed evidence of remobilization) [22]. The micro-injection method is labor intensive however, as transposase mRNA is prepared and embryo injections performed each time remobilization is desired.…”

Effective Expression-Independent Gene Trapping and Mutagenesis Mediated by Sleeping Beauty Transposon

“…SB transposon has been successfully mobilized in mouse somatic cells at frequencies high enough to induce tumors . Tol2 elements were induced to remobilize from their original insertion sites and thus new mutants were generated in zebrafish (Urasaki et al, 2008) and Xenopus (Yergeau et al, 2010), also piggyBac system have been developed successfully for ligand inducible insertional mutagenesis to overcome the adverse effect of the continued expression of transposase (Kong et al, 2010). In this study, we demonstrate that the SB-mediated poly(A)-trap system seems to randomly integrate into intron and exon of target genes and integrated trap cassettes in the HeLa cell genome can be induced to excise from the original insertion site and generate some new integration sites by inducing the expression of SB11 transposase at 37 C. The close-to-random insertion site distribution and remobilization of SB system appears to meet the urgent needs of genome-wide mutagenesis.…”

The Hitchhiker's guide to Xenopus genetics

“…Indeed, this approach has already yielded a dramatic forelimbless phenotype (Abu-Daya et al , 2010) (see below). Remobilization of transposon-based transgenes (Yergeau et al , 2010) shows promise for efficiently generating new insertions, and is compatible with attractive gene-trap and enhancer-trap schemes (Bronchain et al , 1999; Yergeau and Mead, 2007). However, recovery of phenotypes from dedicated insertional screens in X. tropicalis has yet to be reported; whether the rate of gene disruption approaches that of chemical mutagenesis remains a key question.…”