Using putative bovine embryonic stem cell-like cells for nuclear transfer cloning to determine their functional potential

The potential for cloning to improve dissemination of elite genetics has been a goal of livestock scientists for many years. However, efficiency of this technique has remained low compared to other in vitro embryo production methods. One hypothesised method to improve cloning efficiency is to use donor cells that require less reprogramming, as donor cells must be reprogrammed to an embryonic state in order to generate a whole embryo. Somatic cells are the most readily available cell type for donor cells, making them the most often used, but also the most differentiated. Embryonic stem cells (ESCs) are pluripotent cells, capable of generating all cell types. As ESCs are at a much lower differentiated state, they would be hypothesised to improve cloning efficiency. However, ESCs have only been derived from mice and rats. Using a novel culture method, bovine ESC-like cells (ePSCs) can be maintained for several weeks in vitro. The aim of this research was to evaluate the efficiency of using these bovine ePSCs in nuclear transfer (NT) cloning. ePSC colonies were generated from in vitro produced blastocysts and arrested into mitosis with an overnight incubation with 500 nM nocodazole. A method was devised to isolate single cells, using serial treatment in dispase, pronase, and trituration in Ca-Mg-free dissociation media containing cytochalasin B. Arrested colonies were characterised for the degree of mitotic arrest, showing approximately 40% of the colony was arrested in mitosis, and the proportion of cells in DNA synthesis phase had significantly decreased. Cells expressing pluripotency genes SOX2 and NANOG were enriched in the central dome-shaped colony. Analysis of mitotic cells showed that 63% were also pluripotent. NT cloning was optimised for use with donor cells synchronised into mitosis. Pronase treated donor cells fused to metaphase II arrested cytoplasts in isoosmolar fusion buffer resulted in highest fusion rate. Comparison of artificial activation with ionomycin followed by DMAP, CHX, or anisomycin showed that ionomycin/DMAP activation produced the highest blastocyst development rate. Using these optimised conditions, cloning with ePSC donor cells resulted in a three-fold higher blastocyst development than with somatic cells. As donor cells were in metaphase, they contained double the normal amount of DNA. Therefore, it was important to assess the karyotype of blastocysts for normal ploidy. A bimodal distribution of chromosome spreads of around 60 and 120 chromosomes was observed, with higher chromosome numbers apparently restricted to trophoblast cells. Further validation of blastocyst ploidy, characterisation of donor cells used for NT, and continued optimisation of protocols, through double cytoplast cloning or timing of gap between fusion and activation, is required before completing in vivo cloning trials. Embryo transfer trials will be the final step in addressing the long-term aim of this project: to demonstrate that elite bovine embryos can be converted into ePSCs and then ePSC derived animals