ANALYSIS OF THE ROLE OF PRDM14 IN PLURIPOTENCY AND SPECIFICATION OF THE GERM CELL NICHE IN THE DOMESTIC PIG
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The generation of functional, mature germ cells in order to pass on its genetic information to the next generation is of paramount importance to a species’ survival. As a result, primordial germ cells (PGC), the precursor cells that give rise to mature spermatozoa or oocytes, are one of the first cell types to be specified and induced in the early mammalian embryo. In mammals, this process happens during the primitive streak stage of development and PGCs continue to proliferate as they migrate from the posterior end of the embryo through the hindgut and settle in the future gonadal region. One gene in particular, PRDM14, is required for PGC specification in the mouse, but its function was unknown in the domestic pig (Sus scrofa). This dissertation project sought to determine the role of PRDM14 in the porcine germ cell program as well as in early embryo development. The first study used the mouse as a model using CRISPR/Cas9 technology to direct pluripotent stem cells toward the germ cell pathway by aggregation with a host embryo that was unable to form PGCs. Chimeras generated by this technique exhibited 100% occupation of the germline by donor control stem cells. In the second study, mRNA transcripts for a suite of germ cell-related genes were analyzed to determine their expression in various stages of the early porcine embryo: from zygote to blastocyst and embryonic day 28. Unlike in the mouse and human systems, several genes that were predicted to be expressed in the early embryo (PRDM14, TET1, and PRDM1, among others) were found to be extremely low in abundance. In the final study, the function of PRDM14 in the pig was disrupted using gene editing, and the resulting phenotype was characterized. Loss of PRDM14 does not seem to be critical for the specification of PGCs in the domestic pig, as evidenced by the existence of germ cells following knockout. Together, these studies have applications for spermatogonial stem cell transplantation technology in order to expand the genetic reach of superior animals by producing animals whose germ cells are derived from a genetically valuable donor.