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Improving bovine donor cell culture conditions for embryo-derived cell transfer cloning

Cloning by cell transfer offers an efficient method for increasing genetic gain in livestock populations. High-quality genes are routinely induced into embryos with artificial selection-based in vitro fertilisation, or genetic engineering using microinjection. The capacity for large-scale amplification of these superior genes with cell culture is unique to cloning due to its ability to use a single cultured cell as a donor for producing new embryos. Thus, cell transfer cloning could become the primary tool for genetic advancement in the livestock industry if its application is improved. Currently, cell transfer cloning is inefficient in producing viable offspring that survive to term compared to methods such as in vitro production. This is largely caused by epigenetic modifications that arise from the donor cell and the epigenetic abnormalities carried over to the blastocyst generated. Cloning with pluripotent embryonic cells rather than the more widely researched somatic cells may remediate cases of genetic abnormalities. Pluripotent embryo-derived cells are less differentiated than their somatic cell counterparts and can generate cell types from all three of the germ layers, requiring much less reprogramming by the cytoplast. However, the elucidation of optimal conditions to culture pluripotent embryo-derived cells has proved challenging. The primary aim of this research project was to advance methodologies involved in the culture of pluripotent embryo-derived cells in order to increase the efficiency of cell transfer cloning. A series of comparative culture experiments were executed on blastocysts produced through in vitro generation. Experiments focused on the effect of substrate and selection of media components on the generation of an embryo-derived outgrowth. Substrate implementation was found to be essential to initial outgrowth generation as well as further growth. Minimal media with cytokines and small molecules removed was unable to produce outgrowths, indicating the importance of the small molecules and cytokines in attachment, cell proliferation, and survival. Immunofluorescent assays were completed on outgrowths to demonstrate proliferation and cell type markers. Pluripotency marker OCT4 was found in 48% of cell nuclei stained 4 days after plating and in 5% of those stained on day 6. Hypoblast marker Gata6 was not detected in any of the outgrowth cells. The DNA synthesis marker EdU was incorporated into the DNA of 49% of cells after 30 min, which is highly relevant to cloning as donors in S-phase are incompatible with the MII arrested cytoplasts used. A robust protocol was formed for generating outgrowths from bovine blastocysts. Outgrowths demonstrated proliferation and presence of pluripotency markers was illustrated. Cell transfer cloning was completed successfully using embryo-derived donor cells. However, exact donor cell conditions for improved cell transfer efficiency were not elucidated. Further cell transfer experiments should be conducted with a range of donor cell conditions. Karyotyping of the embryos produced would be required before any in vivo implantation of blastocysts into recipients is attempted.
Type of thesis
The University of Waikato
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