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BOR - Papers in Press, published online ahead of print March 24, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028472
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BIOLOGY OF REPRODUCTION 71, 405–409 (2004)
DOI: 10.1095/biolreprod.104.028472
© 2004 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Generation of Transgenic Cattle by Lentiviral Gene Transfer into Oocytes1

Andreas Hofmann3, Valeri Zakhartchenko4, Myriam Weppert4, Heidi Sebald3, Hendrik Wenigerkind4, Gottfried Brem5, Eckhard Wolf4, and Alexander Pfeifer2,3

Department of Pharmacy,3 Institute for Pharmacology, Center for Drug Research, Ludwig-Maximilians University, 81377 Munich, Germany Institute of Molecular Animal Breeding/Gene Center,4 Ludwig-Maximilians University, 81377 Munich, Germany Agrobiogen GmbH,5 86567 Hilgertshausen, Germany

The potential benefits of transgenic cattle range from the production of large quantities of pharmaceutically relevant proteins to agricultural improvement. However, the production of transgenic cattle is presently time-consuming and expensive because of the inefficiency of the classical DNA microinjection technique. Here, we report the use of lentiviruses for the efficient generation of transgenic cattle. Initial attempts to produce transgenic cattle by lentiviral infection of preimplantation embryos were not successful. In contrast, infection of bovine oocytes with lentiviral vectors carrying an enhanced green fluorescent protein (eGFP) expression cassette followed by in vitro fertilization resulted in the birth of transgenic calves. Furthermore, all of the calves generated by infection of oocytes were transgenic, and 100% of these animals expressed eGFP as detected by in vivo imaging and Western blotting. In addition, a transgenic calf was produced by infection of fetal fibroblasts followed by nuclear transfer into enucleated oocytes. Taken together, after adjusting lentiviral transgenesis to cattle, unprecedented high transgenesis and expression rates were achieved.

1 Supported by grants from the Bayerische Forschungsstiftung (492/02) and the DFG.

2 Correspondence: Alexander Pfeifer, Department of Pharmacy, Institute for Pharmacology, Ludwig-Maximilians University, Butenandtstr. 5 (C), 81377 Munich, Germany. FAX: 49 89 2180 77326; alexander.pfeifer{at}cup.uni-muenchen.de




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