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MicroRNA-mediated silencing of bovine NANOG and MBD3

Embryonic stem cells (ESCs) are pluripotent due to their ability to differentiate into any cell type of the body, including functional gametes. These cells are also capable of infinite self-renewal and are highly receptive to genetic modification. The use of ESCs as a reproductive tool offers an exciting platform for farmers to efficiently amplify or introduce desirable agricultural traits within their herd. Unfortunately, all attempts to derive naive pluripotent ESCs from livestock animals have been unsuccessful. This is most likely attributed to a poor understanding of pluripotency specification in these animals. Nanog and Mbd3 are two proteins which are intimately involved in early embryonic development and pluripotency in the mouse. However, their specific function in livestock animals remains unknown. The primary aim of this research project was to determine the feasibility of using a microRNA-mediated silencing approach to knockdown these two proteins in a bovine model. Three candidate microRNAs, designed for each gene transcript, were cloned into the BLOCK-iT™ pcDNA™ 6.2-GW/EmGFP-miR expression plasmid. Before application in an in vivo system, each microRNA was transiently screened in vitro to determine its specificity and knockdown potential. For screening purposes, various attempts were made to generate a stable bovine NANOG expression fibroblast line using an inducible piggyBac transposon expression system. However, stable integration rates were lower than expected and all surviving and overexpressing cell clones eventually entered into a non-proliferate state of cellular senescence. The knockdown potential of the NANOG microRNAs was eventually determined by transient cotransfection with a constitutively active NANOG expression plasmid into a bovine embryonic fibroblast line. All three NANOG-specific microRNAs were able to efficiently knockdown NANOG protein expression. The highest knockdown efficiency was 89% as quantified by immunocytochemistry. By contrast, a MBD3 knockdown effect was not visible after transient expression of the MBD3-specific microRNAs, likely due to the longevity of the MBD3 protein which has a half-life of around 48 hours. The practicality of the BLOCK-iT™ microRNA expression system for functional gene analysis relies on its ability to generate stable knockdown cell lines which could then be used as donor cells for somatic cell nuclear transfer cloning and production of bovine knockdown embryos. Despite the slow proliferative nature of the stable transfectants and their frequent failure to expand during selection, we have been able to eventually generate GFP-microRNA expressing stable knockdown cell clones, suggesting that this system is still a feasible option for investigating the biological functions of NANOG and MBD3 in cattle.
Type of thesis
Wilson, B. C. (2015). MicroRNA-mediated silencing of bovine NANOG and MBD3 (Thesis, Master of Science (Research) (MSc(Research))). University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/9603
University of Waikato
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