Reproductive Technology

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

Abstract
Puromycin N-acetyl transferase gene (pac), whose gene product catalyzes antibiotic puromycin (an effective inhibitor of protein synthesis), has been widely used as a dominant selection marker in ES cell-mediated transgenesis. The present study is the first to report on the usefulness of puromycin for production of 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 prior to somatic cell cloning. The manipulated embryos were transplanted into the oviducts of 14 foster mother sows. Four of the foster sows became pregnant and 9 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 non-cultured cells, and moreover, may confer the production of genetically engineered newborns via nuclear transfer techniques in pigs.

Key words: Embryo • Developmental biology • Early development • Gene regulation

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