42 Disruption of endogenous SOX2 during porcine embryo development using the CRIPSR/Cas9 system

Abstract

The lineage specification of the pre-implantation embryo is important to understand the developmental process, but it remains unclear because the expression of lineage-specific genes is distinct among species. Pigs have genetic and physiological traits similar to humans; however, there are differences in gene expression during the pre-implantation stage. To select a candidate gene that affects the formation of the inner cell mass (ICM) in porcine embryo, we conducted preliminary experiments. First, we measured the expression level of candidate genes for lineage specification in parthenogenetic-activated embryos. The expression of pluripotent genes peaked on Day 3 and thereafter decreased gradually. Next, we conducted immunocytochemistry. OCT4 was expressed in all cells in morula and Day 5 blastocyst, but some Day 7 blastocysts expressed OCT4 in both ICM and trophectoderm (TE), whereas others expressed OCT4 only in ICM. NANOG was not observed in the morula stage, whereas SOX2 was located in a restricted area. To examine the effect of SOX2 in ICM formation, we injected plasmid expressing Cas9 and guide (g)RNA using Lipofectamine for efficient transgene expression at the 2-cell stage to increase viability by inducing mosaicism. The expression of enhanced green fluorescent protein (EGFP) contained in the plasmid confirmed that the plasmid was operating normally. In SOX2-knockout (KO) early blastocysts, the numbers of total cells and SOX2- and NANOG-positive cells were greatly decreased, while OCT4 was expressed in most cells. As in early blastocysts, SOX2-KO late blastocysts had fewer cells expressing SOX2, NANOG, and SOX17 than control. To identify the transcriptional consequences of SOX2 reduction, we performed quantitative PCR analysis on non-injected and PX458-gRNA injected blastocysts. Injection of PX458-gRNA resulted in downregulation of NANOG, SOX17, and SMAD7, but not SOX2 and OCT4. Furthermore, proliferation-associated genes were downregulated in injected blastocysts. In conclusion, SOX2-targeted porcine embryos showed blastocoel formation, the inner cell mass formed poorly, and embryos have inefficient cells. Also, the depletion of SOX2 in porcine blastocysts downregulated pluripotent genes and proliferation genes.

This work was supported by the BK21 Plus Program, the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2019R1C1C1004514), the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the Development of High Value-Added Food Technology Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA; 118042-03-3-HD020).