Role Of CPED1 Gene In Differentiation Of Chicken Embryonic Stem Cells Into Spermatogonial Stem Cells

Spermatogonial stem cells(SSCs)are important adult stem cells in animals,which can transfer genetic material to the next generation,which also can develop embryonic stem cells and develop haploid germ cells.At now a large number of studies have shown that spermatogonial stem cells can be induced in vitro for different functional cell lines for genetic modification,which used to disease treatment and transgenic animal preparation,so the spermatogonial stem cell in vitro operation is particularly important.The formation of spermatozoa in SSCs has been a hot topic for scientists to study,however how to obtain SSCs in large numbers and produce functional sperm has become a scientific problem that scientists need to solve urgently.Based on our lab data of the chicken male ESCs,male PGCs and SSCs RNA-Seq results done previously,we made analysis of the gene expression level in the process of male germ cells,which can help us to discover the unknown genes that have an important role in the process.The CPED1 was highly expressed in the transcriptome of SSCs and could play an important role in the differentiation of SSCs.Therefore,in this study,we used gene overexpression and CRISPR/Cas9 to realize the function of CPED1 of spermatogonial stem cells found in the sequencing process,To provide an efficient method for elucidating the mechanism of germ cell differentiation and differentiation.The main contents of this study are as follows:(1)Based on the pre-RNA-Seq technique,we found that there was a significant difference in the differentiation of CPED1 between ESCs and SSCs.The coding sequence of CPED1 of Rugao yellow chicken was cloned by qPCR,and the expression vector pcDNA3.0-CPED1 and knockdown vector Cas9/gRNA.After transfection of Cas9/gRNA vector with Fugene,the knockout activity of Cas9/gRNA vector were detected by T7E1 digestion,SSA activity detection,TA cloning and off-target efficiency.The activity of Cas9/gRNA1,Cas9/gRNA2 and Cas9/gRNA3 vectors were estimated by fractional gray scale values.The results were 37%,20%and 30%respecitively,Cas9/gRNA1 vector knockout activity was the best;The results of SSA activity showed that the fluorescence activity of gRNAl was about 2 times higher than that of the control group;TA clone sequencing results showed that there were two mutations in the 8 strains of bacteria,and the preliminary knockout rate was about 25%;The results of the off-target efficiency showed that there was no target in DF-1 cells.At the same time,the knockout efficiency of CPED1 in ESCs was 25%.This result indicates that CRISPR/Cas9 technology can stabilize CPED1 knockout on chicken DF-1 and ESCs.(2)The expression of Cas9/gRNA1 and pcDNA3.0-CPED1 were transfected into ESCs by Fugene and after 48 hours changed to RA induction medium.The effect of CPED1 on the differentiation of ESCs into SSCs was detected by cell morphological observation,indirect immunofluorescence assay,flow cytometry and qRT-PCR.The results showed that ESCs knocked out CPED1 could prolong the formation time of embryoid bodies and stop the differentiation of male germ cells compared with normal RA induction group.SSCs were formed in the 12th day and the overexpression group,and no SSCs were found in the knockout group form;The results of indirect immunofluorescence assay showed that the number of integrin ?6 and integrin(31 double-positive type SSCs produced by CPED1 induced 10 days of ESCs differentiation was significantly lower than that of overexpression group and RA induction group;The results of flow cytometry analysis showed that the proportion of integrin a6-positive SSCs in the induction group,overexpression group and knockout group were 1.5%±0.163?1.8%%±0.294 and 0.9%±0.216 respectively.The positive cell rate in the knockout group was significantly lower than that in the other two groups;The expression of CPED1,Cvh,C-kit,Stra8,integrin a6 and integrin ?1 in the RA-induced group were 1.72,1.62,1.74,2.01,2.36 and 2.42 respectively,the expression levels of CPED1,Cvh,C-kit,Stra8,integrin a6 and integrin ?1 in the overexpression group were 2.68,1.71,1.95,2.46,2.67,2.78 respectively,and the expression levels of CPED1,Cvh,C-kit,Stra8,integrin a6 and integrin ?1 in the knockout group were 0.42,1.12,1.03,0.87,1.41,1.53.There was a significant difference in the expression of CPED1 gene between the RA-induced and the overexpression groups,and the expression of CPED1,Cvh,C-kit,Stra8,integrin a6 and integrin ?1 in the knockout group were significantly lower than those in the RA-induced and overexpression groups.In RA-induced differentiation of ESCs into SSCs,knockout of CPED1 inhibited the formation of SSCs,suggesting that CPED1 plays an important role in the regulation of ESCs differentiation into SSCs.(3)The chicks were injected with Cas9/GRNA1 vector and pcDNA3.0-CPED1 overexpressing vector,and the normal hatching group and control group were set up.Respectively,the normal hatching to 4.5d chick embryo reproductive ridge-like and 18.5d testicular samples,using paraffin section and PAS staining,qRT-PCR and flow cytometry for detected the effects of CPED1 knockout and overexpression on the dynamic changes of male germ cells.The expression of Cvh,Stra8 and integrin a6 in the overexpression group of 4.5d were 2.12,1.37,1.20,which Significantly higher than knockout group,and CPED1 expression(1.07)was significantly higher than the knockout group.In 18d the expression level of Nanog in the overexpression group(0.32)was significantly lower than that in the control group,The expression of integrin a6 gene was significantly higher than that of knockout group(1.89),CPED1 expression(2.09)was significantly higher than the knockout group;he results of 4.5d chicken embryo paraffin section showed that the number of PGCs in the control group was(45 ± 2.236),the number of PGCs in the overexpression group was(41 ± 1.699),and the number of PGCs in the knockout group was(18 ± 0.745)The number of PGCs contained in the group was lower than that in the control and overexpression groups;The number of integrin a6 positive cells in the control group,overexpression group and knockout group were 1.3%±0.141,1.6%±0.356 and 0.8%±0.245 respectively,and the number of SSCs in the knockout group was significantly higher than that in the control group Significant reduction in the control group and overexpression group was no significant difference;The results showed that the gene could promote the differentiation of chicken embryonic stem cells into spermatogonia stem cells.