Reproductive Technology

Generation of Cloned Transgenic Cats Expressing Red Fluorescence Protein

Abstract
A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries. Donor fibroblasts were obtained from an ear-skin biopsy of a white male Turkish Angora cat, cultured for one to two passages, and subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein (RFP) gene. A total of 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 ± 7.5/recipient). Three surrogate mothers were successfully impregnated (27.3%), and delivered two liveborn and one stillborn kitten at 65 to 66 days of gestation. Analysis of nine feline specific microsatellite loci confirmed that the cloned cats 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) cat, but not in the surrogate mother cat. Red fluorescence was detected in tissue samples including hair, muscle, brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, tongue and even excrement of the stillborn TG cat. These results suggest that this nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.

Key words: Assisted Reproductive Technology • Developmental biology • Cloned animal • Transgenic animal