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 DNA 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 reliable, and should be considered, in case a single large insert clone of the de...

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Bibliographic Details
Published in:	Transgenic research Vol. 10; no. 3; pp. 211 - 221
Main Authors:	TACKEN, Paul J, VAN DER ZEE, Andre, BEUMER, Tim L, FLORIJN, Ralph J, GIJPELS, Marion J. J, HAVEKES, Louis M, FRANTS, Rune R, VAN DIJK, Ko Willems, HOFKER, Marten H
Format:	Journal Article
Language:	English
Published:	Dordrecht Springer 01-06-2001 Springer Nature B.V
Subjects:	Alternative Splicing Animals Biological and medical sciences Biotechnology Brain - metabolism Deoxyribonucleic acid DNA DNA, Recombinant - administration & dosage DNA, Recombinant - genetics Fundamental and applied biological sciences. Psychology Gene Expression Genetic engineering Genetic technics Humans In Situ Hybridization, Fluorescence Kidney - metabolism lipoprotein (very low density) receptors Low density lipoprotein receptors Male Meiosis Methods. Procedures. Technologies Mice Mice, Transgenic Microinjections Organ Specificity Polymerase Chain Reaction Receptors, LDL - genetics Recombination, Genetic - genetics RNA, Messenger - genetics RNA, Messenger - metabolism Rodents Spermatogenesis - genetics Testis - metabolism Testis - pathology Transgenes - genetics Transgenic animals Transgenic animals and transgenic plants VLDLR gene Lipoprotein VLDL Spermatogenesis Homologous recombination Rodentia Transgenic animal Overlapping sequence Gene expression Testicle Genetic transfer Vertebrata Mammalia Mouse Biological receptor
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Summary:	The generation of functional transgenes via microinjection of overlapping DNA 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 reliable, and should be considered, in case a single large insert clone of the desired 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 VLDLR transgenic (VLDLR-Tg) mice. Three transgenic founders were born, of which 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 resulted 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 complementary 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 parallel the endogenous VLDLR expression, some crucial regulatory elements were probably not present in the cosmid clones. Human VLDLR expression in testis was 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. Furthermore, male VLDLR-Tg mice transmitted the transgene to their progeny with low frequencies. This could imply that VLDLR overexpression in the germ cells disturbed spermatogenesis.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0962-8819 1573-9368
DOI:	10.1023/A:1016682520887