Xenopus transgenics: methods using transposons.

The generation of transgenic animals is an essential tool for many genetic strategies. DNA "cut-and-paste" transposon systems can be used to efficiently modify the Xenopus genome. The DNA transposon substrate, harbored on a circularized plasmid, is co-injected into fertilized Xenopus embryos at the one-cell stage together with mRNA encoding the cognate transposase enzyme.

Forward genetic screens in Xenopus using transposon-mediated insertional mutagenesis.

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.

Remobilization of Sleeping Beauty transposons in the germline of Xenopus tropicalis.

Background: The Sleeping Beauty (SB) transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization.

Transposon transgenesis in Xenopus.

Transposon-mediated integration strategies in Xenopus offer simple and robust methods for the generation of germline transgenic animals. Co-injection of fertilized one-cell embryos with plasmid DNA harboring a transposon transgene and synthetic mRNA encoding the cognate transposase enzyme results in mosaic integration of the transposon at early cleavage stages that are frequently passed through the germline in the adult animal. Micro-injection of fertilized embryos is a routine procedure used by many laboratories that use Xenopus as a developmental model and, as such, the transposon transgenesis method can be performed without additional equipment or specialized methodologies.

Remobilization of Tol2 transposons in Xenopus tropicalis.

Background: The 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.

Transgenesis in Xenopus using the Sleeping Beauty transposon system.

Transposon-based integration systems have been widely used for genetic manipulation of invertebrate and plant model systems. In the past decade, these powerful tools have begun to be used in vertebrates for transgenesis, insertional mutagenesis, and gene therapy applications. Sleeping Beauty (SB) is a member of Tc1/mariner class of transposases and is derived from an inactive form of the gene isolated from Atlantic salmon.