No abstract

Synchronous or asynchronous chimaeras were produced by transplanting a single nucleus of two- and eight-cell embryos from CD-1xCD-1 or BALB/CxBALB/C albino strains into one enucleated blastomere of a late F1 (C57/BLxCBA) x F1 two-cell embryo. The cytoplasmic volume of the blastomere was reduced in some instances by 50%. These chimaeric embryos were cultured in vitro and transferred to pseudopregnant recipients. The distribution of each component to the pups and to the day-10 embryos after transfer to recipients was determined by examining their coat color and by glucose phosphate isomerase analysis, respectively. The contribution of progeny of the nuclear-transplanted cell with nonreduced cytoplast to the pups was 83% when synchronous; this proportion decreased to 43% when asynchronous because the progeny tended to migrate to the trophoblast and/or to the primitive endoderm. When the recipient cytoplast was reduced by 50%, the contribution of the nuclear-transplanted cell progeny to the pups was 79% when synchronous and 80% when asynchronous. This shows that allogenic exchange of a single nucleus at the two-cell stage by nuclear transfer is an effective procedure for producing highly asynchronous mouse chimaeras and suggests that larger and advanced blastomeres tend to be excluded from the inner cell mass of the embryo, but smaller, advanced blastomeres do not.

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...