57 Disruption of TET1 Leads to Abnormal Expression of Pluripotency-Related Genes in Porcine Embryos

DNA methylation is one of the principal epigenetic modifications playing an essential role in regulating gene expression. The TET family (1-3) is implicated in initiating the demethylation process by converting 5-methylcytosine (5mC) to 5-hydroxymethyl cytosine (5hmC) during embryogenesis. Previous studies in mice suggest that TET1 is required for pluripotency and maintenance of embryonic stem cells by managing their epigenetic marks, specifically DNA methylation. This raises the possibility that TET1 is capable of establishing distinct epigenetic marks during embryo development, thus regulating pluripotency-related genes. However, this has not been demonstrated in any species. Previously we have demonstrated that the level of TET1 (mRNA and protein) was high in porcine blastocysts. In this study, we generated TET1 knockout embryos and analysed expression patterns of pluripotency-related genes in blastocysts to study the role of TET1 in maintaining pluripotency during porcine embryo development. The CRISPR/Cas9 system was applied to disrupt the TET1 gene during embryogenesis. Three single-guide RNAs (sgRNAs) were designed based on our previous cloning of the TET1 gene. In vitro-synthesised Cas9 mRNA (20 ng µL−1) and sgRNAs (10 ng µL−1 each) were injected into the cytoplasm of zygotes after IVF. A total of 605 zygotes were used for microinjection and subsequently 54 blastocysts were formed. As a control, 89 IVF blastocysts were developed from 240 embryos. Nine to ten blastocysts per group were collected on Day 7 to analyse gene expression patterns of TET family and pluripotency-related genes using quantitative RT-PCR. Three biological and 3 experimental replications were used. Differences in the gene expression were evaluated by ANOVA. As expected, there was a 2-fold decrease in the transcript level of TET1 in TET1-knockout blastocysts compared with that in control IVF blastocysts (P < 0.05). Interestingly, an increase in TET3 mRNA (P < 0.01) and numeric increase of TET2 mRNA was observed in TET1-knockout blastocysts. We could also detect an elevated level of pluripotency-related genes in TET1-knockout blastocysts; the expression of NANOG, ESRRB, ZFP42, and TCL1A was up-regulated. However, there was no significant change in the expression level of other pluripotency-related genes (POU5F1, SOX2, KLF2, PRDM14, and DPPA3) in TET1 knockout blastocysts. In this study, we found that TET1 is involved in regulating expression of pluripotency-related genes: NANOG, ESRRB, ZFP42, and TCL1A. The loss of functional TET1 resulted in elevated expression of these genes. The reason for this is still under investigation, although TET3, known to have a positive correlation with the level of NANOG, could be involved. A 3-fold increase in TET3 mRNA response to the TET1-knockout may suggest a compensatory mechanism between TET1 and TET3 during porcine embryogenesis. To further understand these actions, we intend to analyse DNA methylation (5mc and 5hmc) levels on the promoter region of the genes. In addition, embryos lacking functional TET1 and TET3 will be generated to explore a potential compensatory effect of TET3 under the absence of TET1.