New genetic techniques by excision of Tol2: isolation of revertants and creating frame shift mutations.

Akihiro Urasaki, Kazuhide Asakawa and Koichi Kawakami

 We have been developing genetic technologies by using the Tol2 transposable element. We have developed transposon-mediated efficient transgenesis, gene trapping, enhancer trapping and jump starter methods in zebrafish. Here we report new applications to manipulate gene functions by using excision of the integrated Tol2 in the genome.
 First, we developed a new method to isolate a revertant. We isolated an insertion of the gene trap construct in an intron of the nup214 gene. Homozygous embryos showed a recessive lethal phenotype. We injected transposase mRNA into the embryos from nup214 heterozygous fish. F1 fish were raised to adulthood and crossed with the nup214 heterozygous fish, and the progeny were analyzed by PCR. Excision of the transposon insertion was identified by performing PCR that can detect the transposon footprint sequence. Thus, we can distinguish the transposon “hit-and-away” allele from the wild type allele. The homozygous fish carrying the “hit-and-away” site developed normally, indicating that the recessive lethal mutation was indeed caused by the transposon insertion.
 Second, we developed a new method to create a frame shift mutation. We isolated an insertion of the enhancer trap construct in an exon of the skiB gene. In this case, since the target duplication contained the GT-AG sequence that can be splice donor and acceptor, an intact mRNA for skiB was synthesized even in homozygous fish. We injected transposase mRNA into the eggs from homozygous fish carrying the skiB insertion, and crossed injected fish with homozygous fish. By performing PCR that can detect a transposon “hit-and-away” allele, we could identify fish that contained a frame shift mutation in the skiB exon.
 These results indicate, when the regulated transpose activity was supplied in vivo, it is possible to control the activity of genes by utilizing excision of existing Tol2 transposon insertions.