Production of Transgenic Pigs Harboring the Human Erythropoietin (hEPO) Gene Using Somatic Cell Nuclear Transfer

Abstract

Abstract. The production of transgenic pigs using somatic cell nuclear transfer (scNT) has been widely described, but a technique for removing nontransfected donor cells and for creating different founder animals has not yet been fully elucidated. In this study, four different expression vectors (pBC1hEPO, pMARBC1hEPO, pBC1hEPOwpre and pMARBC1hEPOwpre) were compared to determine the highest transgene expression, ideal conditions of enrichment of recombinant cells in vitro and efficiency of transgenesis following transfection into HC11 mammary epithelial cells. The highest protein expression in HC11 cells was obtained from the pMARBC1hEPOwpre expression vector. Next, we evaluated the efficiency of transgenic pig production by using geneticin (G418) selection alone or by using real-time PCR selection following G418 selection. Ideal enrichment of recombinant cells was obtained by a combination of realtime PCR and G418 selection; embryos reconstructed using donor cells selected by a combination of real-time PCR and G418 selection gave rise to nine piglets, all of which were transgenic. Among them, three founder transgenic pigs were established. Exogenous DNA fragments were shown to be integrated into chromosomes 1q2.4, 1p2.3 and 6q2.4, respectively, in these three pigs. However, the transgenic rate using G418 selection alone was only 33% (two of six pigs) and showed a very low efficacy compared with that of the combination of real-time PCR and G418 selection. Our results provide a valuable experimental model for applying and evaluating transgenic technology in pigs. Key words: Erythropoietin, Geneticin (G418), Pig, Somatic cell nuclear transfer (SCNT), Transgenic (J. Reprod. Dev. 55: [128][129][130][131][132][133][134][135][136] 2009) enetic manipulation, including transfer of foreign DNA into somatic cells and selection of transgenic somatic cells, is a crucial aspect of producing transgenic animals by somatic cell nuclear transfer (scNT). The low efficiency of scNT livestock production is a major obstacle to widespread use of this technology for production of transgenic animals. Most commercial companies producing transgenic livestock use scNT extensively only to produce transgenic founders. Microinjection of DNA into the pronuclei of fertilized oocytes has been the only practical means of producing transgenic livestock since the method was established in 1985 [1]. However, only a small proportion (approximately 5%) of animals integrate the exogenous DNA into their genome. Many transgenic lines do not provide sufficiently high levels of transgene expression because the integration site is random. Recently, the scNT technique has provided an alternative method for pronuclear microinjection as a means of transferring exogenous DNA to the germ line of an animal that allows for precise genetic modifications by gene targeting [2,3]; Schnieke et al. [2] demonstrated that production of transgenic animals using the scNT technique improved overall efficiency to 2.5 times that of pronuclear microinjection. These previou...

Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer

“…Fibroblast cells were demonstrated to produce clones with a higher efficiency than those of mammary epithelial cells after SCNT (Wilmut et al 1997;Sanchez-Munoz et al 2007). Many transgenic cloned domestic animals have been successfully generated using fetal fibroblasts (Cibelli et al 1998;McCreath et al 2000;Cho et al 2009). Thus, transgenic fetal-derived fibroblast cells were selected for donor cells in our experiments.…”

Germline Transgenic Pigs by Sleeping Beauty Transposition in Porcine Zygotes and Targeted Integration in the Pig Genome

“…Extensive screening of numerous founder animals is currently the only possibility to identify animals with single-copy integrations and the desired expression pattern. The pre-screening of transfected nuclear donor cells in SCNT experiments for transgene and/or selection marker expression is not informative in most cases, since compaction and methylation of genomic DNA change dramatically in cultured cells and the commonly used fibroblasts do not transcribe certain tissue-specific promoters [5], [18].…”

Effects of Cumulus Cells on In Vitro Maturation of Oocytes and Development of Cloned Embryos in the Pig

“…Porcine somatic cell nuclear transfer (SCNT) technology is possibly a useful way to produce valuable animals and has been widely used for basic research (Cho et al. 2009).…”

“…Porcine somatic cell nuclear transfer (SCNT) technology is possibly a useful way to produce valuable animals and has been widely used for basic research (Cho et al 2009). Combined with genetic modification, SCNT technology could also hold huge potential for the use in xenotransplantation or a genetic therapy model for humans (Fujimura et al 2008;Yazaki et al 2009).…”