Study On The Function And Regulation Mechanism Of C2EIP In Chicken Primordial Germ Cells

Primordial germ cells are the progenitor cells of sperms and eggs,which transfer genetic information from the parents to the offspring.This process ensures the stable transmission of genetic material among generations.In recent years,it has gradually become a hotspot in the field of reproductive biology to reveal the molecular mechanism of the key genes in reproductive development by exploring the specific process of primordial germ cell differentiation during the reproductive process.The study of the differentiation of primordial germ cells and the mechanism of its related genes has a great significance such as:(1)The development of animal husbandry,as the study of animal germ's stem cells differentiation can help to solve the problem of Livestock germplasm selection.(2)For human development,where detailed understanding of the reproductive differentiation mechanism can provide a fundamental solution to social problems such as infertility.However,whether it's to solve any aspect of the problem,we need to have a comprehensive understanding of the reproductive process of cell development and genetic mechanisms.However,due to the lack of in-depth exploration of the developmental regulation mechanism of PGCs,it has not been established a mature and inducible culture system in vitro.It is difficult to obtain PGCs with high quantity and high quality,which greatly limits the use of PGCs.Therefore,a comprehensive and in-depth study should be conducted for PGCs development and development mechanism.Based on this,the embryonic stem cells,Primordial germ cells and Spermatogonial stem cells in the normal development in vivo were sequenced and some of the key genes and key signaling pathways that influence PGCs formation were found,and C2EIP(Chromosome 2,Expression In PGCs)gene is one of the most significant differences in the expression of the key genes.The C2EIP gene is highly expressed in PGCs and is not expressed in ESCs and SSCs,suggesting that the gene may be involved in the development and reg?lation of PGCs.In order to study the function of C2EIP gene systematically.In this study,we investigated the function of C2EIP in the process of PGCs formation by using chicken chickens as the research object.Study on the Role of C2EIP Gene by CRISPR/Cas9 Gene Editing Technique.Study the influencing factors of C2EIP expression in different cells.GST-Pull Down,Mass Spectrometry,Immunoprecipitation and other experimental techniques were used to study the molecular regulation mechanism of C2EIP.Our findings can be summarized in the following points:(1)The discovery,cloning and bioinformatics analysis of C2EIP The results of RNA-seq of ESCs,PGCs and SSCs were further analyzed.It showed that C2EIP gene was highly expressed in PGCs and not expressed in ESCs and SSCs.These res?lts were consistent with the results of RT-PCR.The bioinformatics prediction results showed that the C2EIP gene was mainly expressed in the cytoplasm,and it contained phosphorylation and glycosylation sites.Regarding the process of biological evolution,there was a poor conservative among species,but highly conservative in birds.C2EIP possesses the function of ubiquitinated ligase E3 and phosphorylated rapamycin target complex,so that C2EIP has the function of enzyme.(2)The establishment of CRISPR/Cas9 gene editing technique system mediated chicken C2EIP knockout According to the CDS sequence of C2EIP gene provided by NCBI,three gRNA target sites were designed,named gRNA1,gRNA2,and gRNA3,which were ligated into the modified VK001-08 vector.The knockout activity of gRNA3(Value:0.6924±0.07)was twice as high as that of the control group(Value:0.3724±0.12)by SSA activity assay(Value:0.3724±0,12);however,there was no difference between the knockout activity of gRNAl(Value:0.3813 ± 0.27)and gRNA2(Value:0.3718 ± 0.09)at the corresponding target sites and the control group(Value:0.3912±0.32)(no knockout activity,P>0.05).CRISPR/Cas9-gRNA3 was transfected into DF-1 cell with good growth status and DNA was extracted by T7E1 digestion.The knockout efficiency of C2EIP gene was about 27%in DF-1 cells;TA clone sequencing results showed that there were 8 strains of bacteria out of 30 strains showed a different number of base deletions or increased.The gene knockout efficiency was 26%.CRISPR/Cas9-gRNA3 was transfected into ESCs and detected by T7EI digestion.The knockout efficiency was about 20%,at the same time,CRISPR/Cas9-gRNA3 was able to express in the chicken embryo with the highest expression level in the heart,and the gene knockout efficiency was 15%.(3)Research the function of C2EIP during the formation of PGCs in vivo and in vitro We cloned the C2EIP CDS f?ll length,and successf?lly constructed the PCDNA3.0-C2EIP overexpressing vector and C2EIP-N1 fusion expression vector.C2EIP-N1 fusion expression vector was transfected into DF-1,and the expression of green fluorescent protein was observed.It was found that C2EIP gene was not expressed in the cytoplasm.For C2EIP CDS sequences,antigen epitopes were designed to prepare the monoclonal antibodies by synthesis of peptides and intraperitoneal immunization of mice.The antibody titer of the monoclonal antibodies was 1:10,which was detected by IFA,and the antibody antigen response also occurred in the cytoplasm.The function of C2EIP during PGCs formation was studied in vivo and in vitro.In the in-vitro experiments,germ cells were found in the overexpression group after 6 days,and the expression of reproductive markers was significantly expressed(P<0.01);while in knockout group cannot obtain them.The res?lts of flow cytometry showed that the number of Cvh-positive cells decreased(4.8 ± 0.16%)after gene knockout,but increased in the overexpression group(18.6 ± 0.13%).The res?lts of qRT-PCR also showed that the expression of Cvh was significantly down-reg?lated after C2EIP knockout(Value:2.8254±0.31)compared with the normal group(Value:3.1425±0.66,P<0.01).In in-vivo experiments,there was no significant difference in chicken embryo development between overexpression group and control group,but the expression of Cvh gene was significantly increased at day 4.5 in overexpression group than control group and knock out group(P<0.01).Frozen sections of 4.5-day chicken embryos were immunohistochemically tested using Cvh and C-kit specific antibodies.It was shown that the number of PGCs in the knockout group were significantly lower than that in control and overexpression groups.In the normal and overexpression groups,renal development was completely visible,but complete renal structure was not observed in the knockout group.(4)Exploring the factors which influence the differential expression of C2EIP gene The promoter sequence of C2EIP gene was cloned and inserted into EGFP-N1 vector to construct the PC2EIP-EGFP expression vector.EGFP green fluorescence was observed after transient transfection of DF-1,indicating that the promoter region of C2EIP gene has a promoter activity.The PGL3/787(-891?-94bp),PGL3/588(-682?-94bp),PGL3/374(-468?-94bp),PGL3/170(-264?-94bp)vectors were constructed by cloning the different length fragment of the C2EIP gene promoter by 5'end gradually missing technology,and the core region of C2EIP gene promoter was found to be at-264?-94 site by the double luciferase reporter system.STAT1,STAT10,Sox17,Sox2 and Klf5 transcription factor binding sites were found in the promoter core region.Mutation experiments were performed on these sites and the corresponding transcription factor overexpression vector was constructed.It was found that STAT1 co?ld positively reg?late the promoter activity of C2EIP gene by double fluorescein reporter assay;while STAT10,Sox17,Sox2 and Klf5 negatively regulate the C2EIP gene promoter activity.TSA and 5Azacd were added into the culture medium of DF-1 cells which transfected with PGL3/787 vector respectively.The results of the double-fluorescein reporter assay showed that decreasing the level of DNA methylation and improving the level of histone acetylation could increase the C2EIP gene Promoter activity.(5)Researching the molecular regulation mechanisms of C2EIP during the formation of PGCs We constructed C2EIP prokaryotic fusion expression vector.The expression and purification of fusion protein were induced by IPTG.It was shown that C2EIP prokaryotic fusion expression vector could be expressed successfully in prokaryotic expression strains.The interaction proteins of C2EIP gene protein were obtained by GST-Pull Down and mass spectrometry.They were:PTCH2,KRT75,H2B-?,KRT12,Histone H3,SLC25A6,LYZ,VDAC2,Histone H1,EF-1? and KRT19.The accuracy of GST-Pull Down interaction was verified by Western Blot,Co-IP and indirect immunofluorescence.It was confirmed that the C2EIP gene interacted with PTCH2 protein in the endometrium to form complex;The phosphorylation and ubiquitination level of PTCH2 were detected by Co-IP technique in vitro and in vivo.The results showed that high expression of C2EIP could decrease the phosphorylation and ubiquitination level of PTCH2 protein;The shRNA-PTCH2-1,shRNA-PTCH2-2 and shRNA-PTCH2-3 vectors were successfully constructed.At the same time,it was shown that shRNA-PTCH2-3 has a good inhibitory effect on PTCH2 protein.Validation experiments in vivo and in vitro showed that inhibition of HH signaling pathway could inhibit the formation of PGCs(RA:3.8±0.23%;RA+shRNA-IHH:12.9±0.08%;P<0.01).On the contrary,activation of HH signaling pathway could promote the formation of PGCs(RA:3.8 ± 0.23%;RA+shRNA-PTCH2:15.6 ± 0.17%;P<0.01).Combined with the above results,it was found that IHH binds to PTCH2 protein and changes the expression level of PTCH2 active ingredient to regulate the HH signal involved in PGCs formation.C2EIP can also bind to PTCH2 and change the expression of active ingredient of PTCH2 protein by ubiquitination and dephosphorylation,it can be inferred that C2EIP activates the HH signaling pathway through PTCH2 and regulates the formation of PGCs.