[Frontiers in Bioscience 2, d298-308, June 15, 1997]
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TRANSGENIC RABBIT MODELS FOR THE STUDY OF ATHEROSCLEROSIS

John M. Taylor and Jianglin Fan

Gladstone Institute of Cardiovascular Disease, the Department of Physiology, and the Cardiovascular Research Institute, University of California, San Francisco, CA

5. GENERATING TRANSGENIC RABBITS

The first report on the application of transgenic technology to the rabbit was in 1985 by Hammer et al. (34, 35). A construct of the human growth hormone gene directed by the mouse metallothionein promoter was used to investigate transgenic methods in different species by examining construct expression in fetuses and neonates. The transgene was integrated into the rabbit genome with an efficiency of 13%, about half of that observed with the mouse genome. Transgene mRNA was expressed in 25% of the rabbit founders, and one live founder exhibited human growth hormone in serum. In 1988, Knight et al. (36) described the characteristics of three transgenic founders expressing the c-myc oncogene directed by the rabbit immunoglobulin heavy chain enhancer region. These animals were the first reported transgenic rabbits in which transgene expression was characterized. The use of transgenic rabbit blood as a source of transgenic human alpha-1 antitrypsin was described by Massoud et al. in 1990 (37), and a mouse monoclonal antibody gene was used to generate two transgenic rabbits by Weidle et al. in 1991 (38). In 1993, Peng et al. (39) described three interesting transgenic rabbits that were generated with rabbit papillomavirus DNA, in which the transgenic founders developed extensive squamous carcinomas of the skin at an early age.

Fan et al. (13) were the first to apply this technology to the study of lipid metabolism, and they have established several transgenic rabbit models for the study of atherosclerosis. In this protocol (Fig. 1), pathogen-free New Zealand White females are superovulated by an intramuscular administration of follicle-stimulating hormone to enhance the yield of ova, followed 4 days later by intravenous delivery of chorionic gonadotropin. The animals are mated to fertile males, and a typical yield of 20-30 zygotes is flushed from the oviducts 19 hours later. The large male pronucleus of the rabbit zygote is microinjected with a solution of DNA, and then 10-20 injected zygotes are implanted into each oviduct of a recipient female that has been mated previously with a vasectomized male. Following gestation, founder pups are identified by screening DNA using standard techniques when the animals are about one month of age. The microinjection techniques employed in this study were similar to those used for generating transgenic mice with just a few notable differences: 1) rabbit zygotes have about twice the diameter as mouse zygotes but with similar pronuclear sizes; 2) rabbit zygotes have a thick zona pellucida; and 3) rabbit pronuclei appear to be more sensitive to DNA purity.