Optimisation of bovine embryonic stem cell-like line derivation and candidate gene expression analysis

Abstract

The successful derivation of bovine embryonic stem cells (bESCs) has been an area of interest for livestock scientists over the years. These cells possess significant potential for revolutionising livestock breeding by enabling accelerated genetic gain within a single generation. Although robust derivation methods exist for mouse and human ESCs (mESC, hESC), this is not the case for livestock species. Its derivation remains challenging due to the lack of understanding of signalling pathways that maintain pluripotency and regulate early embryo development in livestock species, along with the failure of direct application of mESC and hESC culture conditions. In recent years, studies have reported the development of culture conditions that support bESC derivation, which were adapted in this thesis. This study aims to optimise bESC culture conditions, as well as optimise quantitative PCR methods for gene expression analysis to assess pluripotency. Using cell enrichment treatments, differential plating, colony picking and cell straining, three bESC-like cell lines were derived, which included a wild-type cell line (17), a red fluorescent reporter cell line (R23), and a transgenic β-κ-casein cell line (B2). Although these cell lines were found to express pluripotency markers, both gene and protein expression analysis revealed that the cell lines comprised of a mixed population. Further validation is required to confirm the identity of the derived cells, including using single cell RNA sequencing, karyotyping, in vitro and in vivo differentiation, whole animal generation, and germline contribution assessment.