Doxycycline-inducible overexpression of NANOG in bovine fibroblasts and nuclear transfer embryos
Chibnall, A. M. (2015). Doxycycline-inducible overexpression of NANOG in bovine fibroblasts and nuclear transfer embryos (Thesis, Master of Science (Research) (MSc(Research))). University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/9494
Permanent Research Commons link: https://hdl.handle.net/10289/9494
Naïve pluripotent embryonic stem cells (ES cells) have the ability to give rise to all cell types including functional gametes. These cells have been well established in mice and rats. The potential for biomedical and agricultural applications of pluripotent stem cells in livestock are vast. Having this resource will make in vitro techniques more achievable for research purposes and commercial use. Their use for chimera formation will increase the production of high quality embryos, resulting in a faster inclusion of desirable traits in breeding lines. The aim of this study was to investigate the molecular characteristics of pluripotency in bovine embryos. This is an incremental step to aid our understanding of the greater system which will encourage the derivation of ES cells within the bovine species. An initial target was the key pluripotency transcription factor NANOG. Nanog has thousands of targets and orchestrates the regulation of the naïve pluripotent molecular environment. This thesis will investigate the overexpression of NANOG with a doxycycline-inducible system in bovine female fibroblasts and nuclear transfer (NT) embryos derived from these. It was hypothesised that the overexpression of NANOG would stimulate the pluripotency network, increasing pluripotency. It has previously been recognised that the re-activation of the X-chromosome in females is an indicator of naïve pluripotency in mouse. Notably, the gene Xist which is important for the inactivation of the X-chromosome is directly repressed by Nanog. It was hypothesised that this would hold true in the bovine system. Both the cell line and embryos were analysed for mRNA and protein expression of ectopic NANOG using quantitative PCR (qPCR) and immunocytochemistry. Eight pluripotency-related genes (endogenous NANOG, OCT4, SOX2, KLF4, PDGFRα, SOX17, SOCS3 and FGF4) were quantified using qPCR to investigate the impact of increased NANOG in the NT embryo at day 8. Expression of XIST mRNA was analysed using qPCR and the inactive X-chromosome was identified using immunocytochemistry to understand the role of NANOG in the re activation of the X-chromosome in the bovine system. It was found that the inducible system significantly increased the expression of ectopic NANOG mRNA and protein within the fibroblasts used for NT. A two-fold significant increase of total NANOG mRNA was achieved in the NT embryo, although no additional NANOG protein was identified. The increased NANOG had no effect on the eight pluripotency-related genes investigated. The increased NANOG had no effect on the expression of XIST in the bovine system. No inactive X-chromosomes were identified via immunocytochemistry during this project. Subsequent to this research, details of an alternative overexpression technique using CRISPR technology were published. Use of this system will make research in this field achievable to a higher quality in a shorter timeframe.
University of Waikato
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