Remobilization of integrated transposons: the jump starter system in zebrafish
Akihiro Urasaki, Kazuhide Asakawa and Koichi Kawakami
Division of Molecular and Developmental Biology, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan
We have been developing the gene and enhancer trap methods using the Tol2 transposon, and creating transgenic zebrafish expressing GFP in specific tissues and organs. Currently we are making transposon insertions in the genome by microinjection of both a transposon-donor plasmid and the transposase mRNA into fertilized eggs. Creating a number of zebrafish lines with different transposon insertions would be easier if the jump-starter system, which enables creation of new transposon insertions without microinjection, was developed.
Toward this goal, first we tested whether integrated transposons can be remobilized. We injected transposase mRNA into embryos homozygous for a single transposon insertion in the hoxc3a and the pax6b gene. The integrated transposons were excised from these two loci, and inserted into different loci efficiently. 11 out of 13 reintegration loci mapped on the genome were located on different chromosomes, and two were located on the same chromosome, only 100-kb and 550-kb from the original locus. Thus, integrated transposons can be mobilized by supplying the transposase activity, and, upon remobilization, both genome-wide global hopping and local hopping, predominantly global hopping, were observed. Then, we constructed transgenic fish carrying the transposase cDNA under the control of the zebrafish hsp70 promoter. Double transgenic fish carrying the transposase cDNA and an insertion of the Tol2 enhancer trap construct, causing specific GFP expression, were heat-shocked repeatedly, and their offspring were screened for new patterns during embryonic development. We found that the germ cells of the heat-shocked fish became highly mosaic, and a pair of the male and the female could generate more than 11 new patterns at the frequency of approximately one pattern per 20 embryos. Southern blot analysis and inverse PCR revealed that these new GFP expression patterns indeed resulted from transposon insertions at new loci. In these cases, global hopping was also predominant. These results indicate that the jump starter system we established here can be used to create transposon insertions in new loci efficiently in the germ cells.