58 Handmade Cloning in Trans-Species Nt: Culture Medium Has an Effect on the Ability of Reconstructed Bovine-Murine Embryos to Develop Beyond the 8-Cell Stage

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...

Uso de oócitos bovinos como citoplasma receptor na produção de embriões por transferência nuclear de célula somática interespécie (NTSCi)

“…They compared the in vitro development of NTSCi murinesbovine embryos maintained into an adequate culture medium to promote the development of murines embryos (KSOM) with embryos that were cultivated in a means usually used for the culture of bovine embryos (SOF). The system that used KSOM medium showed better in vitro development rates and provided the development up to the morula stage, even though none of them reached the blastocyst stage [20]. In this way, the system of in vitro the culture seems to be directly related to the success of the NTSCi and the fact that we achieved the development up to the blastocyst of NTSCi caprine-bovine embryos shows that this is possible.…”

Analysis of mice (Mus Musculus L.) and hamster embryo development using culture and vitrification medium: Systematic review

“…During cytoplasmic maturation, molecular and structural changes occur, namely a rapid increase in the number and size of organelles such as ribosomes, fat globules, Golgi apparatus, and mitochondria. Based on the research that has been done, it has been proven that the KSOMaa medium is able to stimulate the growth and development of the embryo at the stage of 8-cell division in the aggregation process so that it can produce aggregated embryos with high viability ( Nieminen et al , 2004 ).…”