Somatic nuclear transfer has been a useful technique for the production of genetically engineered domestic animals including gene-targeted and transgenic animals. To date, transgenic sheep, pigs, and cattle and gene-targeted sheep and pigs have been successfully produced by nuclear transfer employing transfected somatic cells as nuclear donors. These genetically defined cloned animals may be used for xenotransplantation as a source for human therapeutics. However, production of these animals is time consuming and requires significant effort because it involves a lengthy protocol comprising several steps that include the isolation of somatic cells from adult animals (or fetuses), cultivation of these cells as a proliferative cell line, characterization of cells to be used for somatic cell cloning, and finally selection of cells with G418 after transfection. This is particularly evident when we consider selection with G418, as it requires high concentrations of G418 (more than 100 ^g/ml), and a long selection period under culture (around 10 days), which occasionally causes alteration in cell potentiality and cy-tological aging. In fact, cells cultivated for long periods of time are associated with poor-quality cell nuclei in the simple nuclear transfer system. It is highly desirable to shorten as much as possible the cell culture periods required for isolation of somatic cells, gene transfer, and drug screening of recombinants before nuclear transfer.
Puromycin is an antibiotic that inhibits growth of animal cells and blocks protein synthesis by binding to 80S ribosomes at low doses. The puromycin-resistant gene (termed pac) encoding puromycin Af-acetyl transferase was isolated from Streptomyces aboniger. If pac is introduced and expressed in animal cells, the cells can survive in the presence of puromycin. Our previous report demonstrated that puromycin killed wild-type embryonic stem (ES) cells after short periods of exposure (2 days) in mice. Furthermore, puromycin was effective at a very low concentration of 1 ^g/ml. It generally takes only 7 days to obtain stable transfectants. On the other hand, G418 has been widely used for the selection of various cells after transfection. In stark contrast with puromycin, G418 requires 10-14 days to obtain transfectants and much higher concentrations (100-1500 ^g/ml) are required to kill nontransduced cells.
In this study, we investigated effects of puromycin for the selection of transduced cells to use in porcine nuclear transfer experiments. Somatic cells primarily isolated from porcine fetuses were directly subjected to electroporation with an enhanced green fluorescent protein (EGFP) expression vector containing pac. These electroporated cells then underwent selection with puromycin for 7 days. After selection, EGFP-expressing transfectants were directly subjected to nuclear transfer in pigs. As a result, we obtained one healthy piglet expressing EGFP systemically. The significance of our simplified method for generating transgenic pigs via nuclear transfer is discussed.