Effective generation of very low density lipoprotein receptor transgenic mice by overlapping genomic DNA fragments: high testis expression and disturbed spermatogenesis

The generation of functional transgenes via microinjection of overlapping D NA fragments has previously been reported to be successful, but it is still not a widely applied approach. Here we show that the method is very reliab le, and should be considered, in case a single large insert clone of the de sired gene is not available. In the present study, two large DNA fragments consisting of overlapping cosmids, together constituting the human very low density lipoprotein receptor (VLDLR) gene (35 kb), were used to generate V LDLR transgenic (VLDLR-Tg) mice. Three transgenic founders were born, of wh ich two (strain #2 and #3) generated transgenic offspring. Using Fiber-FISH analysis, the integration site was shown to contain at least 44 and 64 DNA fragments in mouse strains #2 and #3, respectively. This copy number resul ted in integration sites of 1.5 and 2.5 megabase in size. Notably, over 90% of the fragments in both mouse strains #2 and #3 were flanked by their com plementary fragment. In line with this observation, Southern blot analysis demonstrated that the correct recombination between fragments predominated in the transgenic insertion. Human VLDLR expression was detected in testis, kidney and brain of both mouse strains. Since this pattern did not paralle l the endogenous VLDLR expression, some crucial regulatory elements were pr obably not present in the cosmid clones. Human VLDLR expression in testis w as detected in germ cells up to the meiotic stage by in situ mRNA analysis. Remarkably, in the F1 generation of both VLDLR-Tg mouse strains the testis was atrophic and giant cells were detected in the semineferous tubuli. Fur thermore, male VLDLR-Tg mice transmitted the transgene to their progeny wit h low frequencies. This could imply that VLDLR overexpression in the germ c ells disturbed spermatogenesis.