Investigation of CDX2 role in trophectoderm differentiation of bovine preimplantation embryos by using CRİSPR/CASs9

While detailed studies have been conducted on cell lineage differentiation during the zygote-to-blastoscsyt formation process in mice as a model mammalian species, this process remains uncertain in bovine. Moreover, early pregnancy losses frequently occur in bovine embryos produced using assisted reproductive techniques, making it an ongoing issue that needs to be addressed. It is anticipated that a better understanding of the factors that effective on the differentiation of trophectoderm (TE) and inner cell mass (ICM) during preimplantation development could help prevent early pregnancy losses. In this context, it was aimed to investigate the role of the caudal type homeobox transcription factor-2 (CDX2) gene in bovine, which is known to be involved in the preimplantation development process of bovine and is required for the differentiation of TE/ICM lineages in mouse embryos. For this purpose, embryos with CDX2 gene silenced (knock-out) using CRISPR/Cas9 technology were produced in vitro and the effect of silencing the CDX2 gene on bovine preimplantation development was monitored. The study material consisted of oocytes obtained from ovaries collected from slaughterhouses. Bovine oocytes were injected with mRNA encoding only Cas9 to create a control group, and with CDX2-targeted sgRNA+Cas9 mRNA to form the experimental group. Bovine blastocysts on the 8th day of preimplantation development were fixed and embryo morphology was examined by image analysis for CDX2, GATA3 and SOX2 immunohistochemical staining to identify TE/ICM cells, respectively. DNA isolation was conducted to identify which embryos had successfully silenced the CDX2 gene, and genotyping was performed using the MiSeq technology. As a result of all the analyses, it has been determined that there is no statistically significant difference in embryo development with respect to the CDX2 gene between the control and experimental groups. The information obtained in this thesis study, when compared to mouse embryos, indicates that CDX2 does not play a vital role in trophectoderm differentiation and blastocyst formation in bovine embryos. Furthermore, it has been found that the first cell lineage differentiation during embryo development differs among mammalian species. Based on these results, it can be suggested that the investigation of all genes presumed to be involved in the losses observed in bovine embryos produced using assisted reproductive techniques is necessary to determine the causes.