63 PRODUCTION OF CLONED TRANSGENIC RED CATS

Abstract

A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Because the feline gene map displays a higher level of systemic conservation with the human genome than with rodents or other laboratory mammals, cats may offer a more appropriate model in which to study potential therapies for human diseases. A second point of application is the production of transgenic cats, genetically engineered designer pets with favorable attributes, such as allergen-free cats. Mature oocytes from superovulated cat ovaries were collected. Donor fibroblasts were obtained from an ear-skin biopsy of a white maleTurkish Angora cat, cultured for one to two passages, and then subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein (RFP) gene. Single donor cells emitting red fluorescence were selected under an inverted microscope equipped with an RFP filter set. For somatic cell nuclear transfer (SCNT), denuded oocytes were treated with 0.2 µg m L^–1 demecolcine for 1 h, and the protruding first polar body and chromatin plate were removed by micromanipulation. A single donor cell was transferred into each enucleated oocyte. In total, 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 ± 7.5 per recipient). Three of eleven surrogate mothers (S) were successfully impregnated (27.3%). Surrogate S-2 delivered one liveborn and one stillborn kitten at 65 days of gestation while S-3 delivered one liveborn kitten at 66 days of gestation. Microsatellite analysis revealed that all kittens were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cats, but not in the non-TG cats under excitation emission filter sets. The expression pattern of the RFP transgene was determined in several tissues taken from TG stillborn and non-TG cats. Although expression in the heart was detected more weakly than in other organs, red fluorescence was present in all tissue samples tested, but not in those from the non-TG cat. This is the first report of the successful production of a cloned transgenic cat expressing an exogenous gene. Our nuclear transfer procedure using genetically modified somatic cells would be useful for the efficient production of transgenic cats. The application of the SCNT procedure to produce genetically modified cats would be valuable for biomedical modeling of human disease, as well as for the production of designer pets.

This work was supported by a Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MOST) (grant # M10525010001-05N2501-00110).