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A Novel Method for the Production of Transgenic Cloned Pigs: Electroporation-Mediated Gene Transfer to Non-Cultured Cells and Subsequent Selection with Puromycin¹

Department of Developmental Biology,⁴ Division of Insect and Animal, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-0901, Japan Prime Tech Ltd.,⁵ Tsuchiura, Ibaraki, 300-841, Japan The Institute of Medical Sciences,⁶ Tokai University, Bohseidai, Isehara, Kanagawa, 259-1143, Japan

Puromycin N-acetyl transferase gene (pac), of which the gene product catalyzes antibiotic puromycin (an effective inhibitor of protein synthesis), has been widely used as a dominant selection marker in embryonic stem (ES) cell-mediated transgenesis. The present study is the first to report on the usefulness of puromycin for production of enhanced green fluorescent protein (EGFP) transgenic piglets after somatic cell cloning and embryo transfer. Somatic cells isolated from porcine fetuses at 73 days of gestation were immediately electroporated with a transgene (pCAG-EGFPac) carrying both EGFP cDNA and pac. This procedure aims to avoid aging effects thought to be generated during cell culture. The recombinant cells were selected with puromycin at a low concentration (2 µg/ml), cultured for 7 days, and then screened for EGFP expression before somatic cell cloning. The manipulated embryos were transplanted into the oviducts of 14 foster mother sows. Four of the foster sows became pregnant and nine piglets were delivered. Of the nine piglets, eight died shortly after birth and one grew healthy after weaning. Results indicate that puromycin can be used for the selection of recombinant cells from noncultured cells, and moreover, may confer the production of genetically engineered newborns via nuclear transfer techniques in pigs.

¹ Supported by a grant for Establishment of Transgenic Agro-Biofarm Systems from the Ministry of Agriculture, Forestry and Fisheries of Japan.

² Correspondence: Satoshi Watanabe, Department of Developmental Biology, Division of Insect and Animal, National Institute of Agrobiological Sciences, Ikenodai 2, Tsukuba, Ibaraki, 305-0901, Japan. FAX: 81 29 838 8635; kettle{at}affrc.go.jp

³ Current address: Department of Research Planning and Coordination, National Institute of Livestock and Grassland Science, 2 Ikenodai, Tsukuba, Ibaraki, 305-0901, Japan