Study On The Role Of Candidate Gene LBC In The Formation Of Chicken Spermatogonial Stem Cells

Spermatogonial stem cells(SSCs),the only stem cell in male adult stem cells,can transmit genetic material to the next generation of stem cells,SSCs is the reproductive stem cells,and it is the basis of spermatogenesis.In the study of spermatogenesis,Disease and treatment of male infertility and other fields,it have an irreplaceable role.The formation of spermatogonial stem cells is regulated by related genes,but its specific molecular mechanism is now unknown.In this study,we studied the specific expression of new gene LBCs on spermatogonial stem cells.Rugao yellow chicken was used as experimental animals in vitro and in vivo In addition to overexpression of LBC gene,the role of LBC in the formation of chicken stem cells was studied,which provided a reference for clarifying the mechanism of differentiation and differentiation of spermatogonial stem cells.1.Cloning and sequence analysis of LBC.We used Nest PCR to clone LBC gene CDS,and inserted it to pMD19-T vector.Restriction enzyme digestion and sequencing confirmed that the recombinant vector pMD 19-T-LBC was correctly constructed,Sequence alignment showed its homology with LBC cDNA of chicken from GenBank is 99.43%.There are two changes in the number of amino acids encoded,the successful cloning of Rugao yellow chicken LBC gene,can be used for follow-up study.2.Preparation and subcellular localization of polyclonal antibody against LBC gene.The antigen of the LBC gene was prepared and the mice were immunized.The serum was collected and the antibody titer was detected by indirect immunofluorescence(IFA).The eukaryotic expression vector pEGFP-Nl-LBC was constructed and transfected into DF-1 cells for 48 h.The transfection results were observed under fluorescence uptake microscope.The RT-PCR and indirect immunofluorescence assay were used to further identify LBC-eGFP mRNA and protein Expression state.The results showed that the expression of fusion protein was detected by RT-PCR.The results of fluorescence microscopy and IFA showed that LBC-eGFP fusion protein was expressed in the nucleus and cytoplasm,which was consistent with the results of bioinformatics prediction.3.Effects of LBC gene on spermatogonial stem cell formation.Construction of LBC knockout vector CRISPR/Cas 9-LB C and overexpression vector pcDNA3.0-LBC.After transfection of DF-1 cells,the vector activity was verified by sequencing,T7E1 digestion and Western Blot.The plasmids were transfected into ESC and cultured in RA culture medium.Morphological observation,immunochemical detection and QRT-PCR were used to detect the effect of LBC deletion and overexpression on SSC differentiation in vitro.The effects of LBC deletion and overexpression on the formation of PGCs and SSCs were detected by paraffin section and flow cytometry.The results showed that both CRISPR/Cas9-LBC and pcDNA3.0-LBC vectors were constructed successfully and active.In vitro experiments,compared with RA induction group,LBC overexpression significantly promoted the differentiation of ESC to SSC,and the expression of integrin a6 and integrin ?1 in SSCs increased significantly.In contrast,the differentiation of ESCs into SSCs was inhibited to a certain extent in the case of LBC gene knockout.Clones formed on day 6 but did not form spermatogonial cells.The expression of Sox2 in the ESCs was inhibited and the expression of integrin a6 and integrin ?1 was also decreased.In vivo experiments,LBC gene overexpression significantly promoted the formation of PGC and SSC cells compared with normal developed chicken embryos,whereas LBC knockout significantly reduced the formation of PGC and SSC cells.