Transgenesis and Nuclear Transfer Using Porcine Embryonic Germ Cells

Abstract

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). These cells share many characteristics with embryonic stem cells including their morphology and pluripotency. Undifferentiated porcine EG cell lines demonstrating capacities of both in vitro and in vivo differentiation have been established (Shim H et al. 1997 Biol. Reprod. 57, 1089-1095). Since EG cells can be cultured indefinitely in an undifferentiated state, whereas somatic cells in primary culture are often unstable and have limited lifespan, EG cells may provide an inexhaustible source of karyoplasts in nuclear transfer (NT). This would be particularly advantageous in maintaining nuclear donor cells carrying a transgene. In addition, genome-wide demethylation of DNA occurs in pre-implantation embryos as well as PGC. Nuclear transfer embryos using EG cells rather than somatic cells may be close to embryos from normal fertilization in their DNA methylation status. If combined with NT technique, EG cells may potentially be useful for genetic manipulation in pigs. In this study the efficiencies of transgenesis and NT using porcine fetal fibroblast and EG cells were compared. Two different techniques were used to perform NT. When conventional NT procedure (Roslin method) involving fusion of donor cells with enucleated oocytes was used, the rates of development to the blastocyst stage were 16.8% (59/351) and 14.1% (50/354) in EG and somatic cell NT, respectively. In piezo-driven micromanipulation (Honolulu method) involving direct injection of donor nuclei into enucleated oocytes, the rates of blastocyst formation in EG and somatic cell NT were 11.9% (15/126) and 7.5% (12/160), respectively. Although the differences between EG and somatic cell NT were statistically insignificant, the rates of blastocyst development in EG cell NT were comparable to the somatic cell counterpart regardless of NT methods used in the present study. To investigate if EG cells can be used for transgenesis in pigs, GFP gene was introduced into porcine EG cells. Nuclear transfer embryos using transfected EG cells gave rise to blastocysts (29/137, 21.2%), and all embryos that developed to the blastocyst stage expressed GFP, based on observation under fluorescence microscope. In this study, the possibility of using EG cells as karyoplast donors in NT procedure was tested. The results suggest that EG cell NT may be used as an alternative to somatic cell NT, and transgenic pig embryos may be produced using EG cells. It has been reported that aggregation of two nuclear transfer (NT) mouse embryos shows an improvement in full-term development (Boiani M et al. 2003 EMBO J. 22, 5304-5312). In this study, we examined the effect of aggregation on in vitro development of bovine NT embryos. As donor cells for NT, cumulus cells of passage 3-5 were used following culture in serum-starved medium for 5-7 days. NT was performed as previously described (Akagi S et al. 2003 Mol. Reprod. Dev. 66, 264-272). NT embryos were cultured in a serum-free me...

Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer: multi-factorial analysis of a large data set

“…However, this does not seem to be a general feature of mesenchymal stem cells, since in more recent cloning experiments with adipose tissue derived mesenchymal stem cells a high proportion of viable offspring without malformations was obtained (T. Flisikowska and A. Schnieke, unpublished data). Some groups reported that mesenchymal stem cells are superior to fibroblasts for SCNT in pigs [50-53], although this has not been generally observed [40,54,55]. Our results did not show any differences in the cloning efficiency among the different cell sources tested, although there was a tendency for a higher pregnancy rate when mesenchymal stem cells were used.…”

“…Generally, genetic modification of donor cells requires prolonged in vitro culture for transfection and selection, which could induce cellular changes leading to a decrease in cloning efficiency. Gene targeting by homologous recombination takes a particularly long time and multiple cell divisions to establish single cell clones with sufficient cell numbers for genetic analysis and nuclear transfer [5,38-40]. In contrast, our protocol for additive gene transfer uses pools of mixed cell clones, which have been maintained under selection for 7 to 10 days [8,18,41].…”

39 Factors Influencing the Efficiency of Generating Genetically Engineered Pigs by Nuclear Transfer: Multi-Factorial Analysis of a Large Data Set

“…However, this does not seem to be a general feature of mesenchymal stem cells, since in more recent cloning experiments with adipose tissue derived mesenchymal stem cells a high proportion of viable offspring without malformations was obtained (T. Flisikowska and A. Schnieke, unpublished data). Some groups reported that mesenchymal stem cells are superior to fibroblasts for SCNT in pigs [50][51][52][53], although this has not been generally observed [40,54,55]. Our results did not show any differences in the cloning efficiency among the different cell sources tested, although there was a tendency for a higher pregnancy rate when mesenchymal stem cells were used.…”

“…Generally, genetic modification of donor cells requires prolonged in vitro culture for transfection and selection, which could induce cellular changes leading to a decrease in cloning efficiency. Gene targeting by homologous recombination takes a particularly long time and multiple cell divisions to establish single cell clones with sufficient cell numbers for genetic analysis and nuclear transfer [5,[38][39][40]. In contrast, our protocol for additive gene transfer uses pools of mixed cell clones, which have been maintained under selection for 7 to 10 days No selection was used as the reference category.…”

Pluripotent cells in farm animals: state of the art and future perspectives

“…Recent in-depth characterisation of putative porcine EGCs by functional and molecular studies, including RNA sequencing (RNAseq), indicates that the cells are rather multipotent than pluripotent and do not show higher expression levels of some of the key pluripotency transcription factors than porcine embryonic fibroblasts (Petkov et al 2011). EGCs can potentially be cultured in vitro indefinitely in the undifferentiated state, and thus have the potential to overcome the limitations encountered with the short lifespan of somatic cells and to facilitate the production of transgenic animals (Rui et al 2004(Rui et al , 2006Ahn et al 2007). Porcine EGCs were stably transfected with an enhanced green fluorescent protein (EGFP) construct (Piedrahita et al 1998;Rui et al 2006;Ahn et al 2007) and EGFP-positive EGCs integrated into the inner cell mass of the host blastocyst after injection (Rui et al 2006) or gave rise to blastocysts expressing EGFP after use as donor cells in somatic cell nuclear transfer (Ahn et al 2007).…”