Stable and silencing-free expression of an eGFP transgene in ES cells and mice generated by cassette exchange method

摘要

Recombinase-mediated cassette exchange (RMCE), with which a single copy of a transgene can be inserted into a chosen locus by a site-specific recombinase, provides a number of potential merits over conventional transgenic (Tg) methods. Here, we developed a novel selection marker-free integration system, and generated eGFP Tg mice using this method. We first established ES cell lines, which contain an exchangeable cassette at specified loci (ROSA26 and H2-Tw3). We next integrated a cassette containing CAGGS-EGFP-pA, a promoter-less lacZ, and hygromycin selection genes into these loci by Cre-based RMCE. The 20.6-38.5% of hygromycin resistant ES colonies were the correct RMCE clones that were easily distinguishable from randomly integrated ones by lacZ expression. Expression of the eGFP gene, however, appeared to be under epigenetic silencing in both ES cell and mouse derived from RMCE clone, since it showed evident mosaicism. We administrated FLPe expression plasmid to remove the selectable marker and prokaryotic vector sequence. As a result, ES cells and mice without these sequences exhibited very bright and even EGFP fluorescence. Our system promises the targeted integration of a gene of interest for stable expression.