Abstract
In an insect cell line BmN4 derived from Bombyx mori, foreign genes were efficiently integrated in chromosomal DNA by the piggyBac transposition system composed of the donor plasmid harboring the piggyBac element and the helper plasmid expressing the transposase gene. An analysis of transformed cells, however, revealed that the presence of mRNA and activity of the transposase for more than 50 days after the co-transfection of the plasmids, suggesting the re-mobilization of the integrated piggyBac element could be caused by a continuous production of active transposase. To shorten the period of transposase production, four transposase mRNA constructs with different untranslated region (UTR) sequences were synthesized in vitro and used as alternatives for the helper plasmid. One mRNA construct, whose 5’ and 3’ UTR sequences except for the polyA tail were similar to those of the mature transcript expressed from the helper plasmid, was effective in the transposition of the piggyBac element from the co-transfected donor plasmid into the genome and was comparable to the helper plasmid in the efficiency of its transformation. In addition, the transfected mRNA disappeared earlier than the transcript from the helper plasmid and the transposase activity became undetectable after around 21 days of post-transfection during the antibiotic selection. The results clearly indicated that, as a source of transposase for the piggyBac-mediated transformation of BmN4 cells, and possibly other cultured cells and living organisms as well, the in vitro synthesized mRNA with the appropriate 5’ and 3’ UTR sequences is superior in the stability of the integrated piggyBac element to the conventional helper plasmid.
© 2011 by Japan Academic Association for Copyright Clearance (Except in the USA), Copyright Clearance Center, Inc. (In the USA)
