C1EIS Regulates Chicken ESC Differentiation Into Male Germ Cells

Endogenous factors and exogenous factors controled the fate of ESC to SSC. RA, BMP4 exogenous induction and Stra8, DAZL endogenous genes regulation formed regulatory networks to performed regulation function. When differentiation mechanism were explored, novel genes were found specifically expressed in germ cells. Exploring the gene function represented profound values for ESC induction system and diseases of the reproductive. The research realized gene knockout in chicken by CRISPR/Cas9 genome editing technology and found C1EIS gene function in SSC differentiation process. The research offered useful information for optimized induction conditions of ESCs to germ cells, with the promise of facilitating our understanding of mechanisms for cytogenesis, proliferation and differentiation of germ cells. The results addressed as follows:1 We previously found C1EIS expression was significantly different in the process of ESC to SSC. The full-length cDNA of C1EIS was cloned by qPCR, following sequenceing. bioinformatics analysis results showed that C1EIS gene（GenBank accession No. XM₄16004.2） encode 144 amino acid. The protein was instable lipid soluble protein without signal peptide. C1EIS protein contain 2 O-glycosylation sites and IN-glycosylation sites. The secondary structure of PPAR a contains alpha helix, beta turn, and extended strand, random coil, phylogenetic tree analysis of C1EIS showed that the nearest relationship existed between gallus and anas platyrhynchos among all the species mentioned above, and the lowest homology was bos taurus.2 We designed 3 sgRNA sequence for structuring CRISPR/Cas9 vector. SgRNA sequence were inserted into CRISPR/Cas9 knockout vector. We performed a T7EI mutagenesis assay and DNA sequencing to verify the CRISPR/Cas9 vector’s efficiency, which indicated approximately 20% of chicken embryo fibroblasts were successfully mutagenized. CRISPR/Cas9 technology is a robust tool that accomplish targeting gene mutagenized in chicken cells. We amplified C1EIS for structuring pcDNA3.0 overexpression vector The activity was identified by EcoR I and Kpn I enzyme digestion, which indicated the expression vector is successfully constructed.3 ESC were transfected with CRISPR/Cas9 and pcDNA3.Q-C1EIS vector for 48h, subsequently induced with RA for 12d. We detected the differentiation of the targeted ESCs into SSCs by morphology, immunocytochemistry, flow cytometry and Q-RT-PCR. EBs significantly decreased 75% and quit to differentiate into SSC in 6d, SSC-like cells amount distinctly reduced in 12d, when compared with control group. Immunofluorescent staining revealed that the mutagenized ESCs expressed lower levels of integrins α6 and β1 compared to wild type cells. Flow analysis revealed mutation group integrins α6 and β1 positive cells amount is 0.1%, when control group and overexpression group was 20.8% and 19.7%. Q-RT-PCR revealed Sox2 and Nanog expression significantly increased 51% and 42%, contrarily integrin β1 and Stra8 expression significantly decreased 72% and 38% than RA induced group. During retinoic acid-induced differentiation, knockout of C1EIS in ESCs inhibited formation of SSC-like cells, suggesting CIEIS plays a vital role in promoting differentiation of avian ESCs to SSCs by regulating expression of multiple pluripotency-related genes.