PuromycinÂ N-acetyl transferase gene (pac), of which the gene product catalyzes antibioticÂ puromycin, an effectiveÂ inhibitorÂ ofÂ 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 ofÂ puromycinÂ 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 andÂ pac.Â 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 Î¼g/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.
Fig 1.Â EGFP expression in porcine fetal somatic cells (A,Â B) surviving in the presence of 2 Î¼g/ml ofÂ puromycinÂ for 7 days after transfection with EGFPac and in blastocysts (C,Â D) derived from embryos transferred with nuclei of the EGFP-expressing cells. Cells and blastocysts were microscopically inspected for EGFP fluorescence under UV illumination. Note that almost all cells in transfected fetal somatic cells and nuclear transplanted blastocysts expressed EGFP strongly.Â AÂ andÂ C, bright field;Â BÂ andÂ D, dark field
Puromycin is anÂ antibioticÂ that inhibits growth of animal cells and blocks protein synthesis by binding to 80S ribosomes at low doses. TheÂ puromycin-resistant geneÂ (termedÂ pac) encodingÂ puromycinÂ N-acetyl transferaseÂ was isolated fromÂ Streptomyces aboniger. IfÂ pacÂ is introduced and expressed in animal cells, the cells can survive in the presence of puromycin. Our previous reportÂ 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 Î¼g/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 10â€“14 days to obtain transfectants and much higher concentrations (100â€“1500 Î¼g/ml)Â are required to kill nontransduced cells.
PUROMYCIN SENSITIVITY ASSAY FOR FETAL PORCINE SOMATIC CELLS
To determine the optimal concentration of puromycin for the selection of pac-transfected cells, aÂ puromycin resistance testwas performed by culturing fetal porcine somatic cells in a medium containing 0.5â€“6.0 Î¼g/ml of puromycin for 7 days. No surviving colonies were obtained when exposed to puromycin at concentrations higher than 2 Î¼g/ml. When cells were grown in medium containing 2 Î¼g/ml of puromycin, none of the resultant cells survived for more than 6 days. We therefore used 2 Î¼g/ml of puromycin for the selection of cells transfected with EGFPac.
ISOLATION OF PUROMYCIN-RESISTANT PIG SOMATIC CELLS
Approximately 10Â cells survived in the presence of puromycin (2 Î¼g/ml) after gene transfer involving the EGFPac construct. Almost all of these surviving cells exhibited EGFP fluorescence (Fig. 1, A and B). A portion of cells (a total of eight colonies) that had survived after selection with puromycin was then examined for the presence of EGFP cDNA by PCR; the EGFP cDNA was detected in each sample (data not shown). Western blotting analysis using an anti-GFP antibody also confirmed that these surviving cells expressed EGFP protein (which was identified as a 38-kDa band).
SOURCES:Â Satoshi Watanabe, Masaki Iwamoto, Shun-ichi Suzuki, Daiichiro Fuchimoto, Daisuke Honma, Takashi Nagai, Michiko Hashimoto, Satoko Yazaki, Masahiro Sato and Akira Onishi