Screening And Verification Of Key Factors By High-throughput In Dairy Goat Male Germline Stem Cells

Spermatogonial stem cells(SSCs), also names as Male Germline Stem Cells(mGSCs).Spermatogonial stem cells(SSCs)are a kind of adult stem cells which could maintain their population stability by self-renewal and differentiate, and locatd in base membrane of seminiferous tubule in testis,. SSCs are not only retain the characteristics of stem cells but also the features in male germline cells. SSCs are an suitable cellular model for the reach of spermatogenesis, meiosis and cellular reprogramming in vitro. Spermatogenesis is a complicated biological process, effected by mass of regulated factors. These factors are divided to two types, one are coding-RNAs, and other are noncoding-RNA, such as miRNAs,piRNAs and lnc RNAs.However, the transcripts profiling in dairy goat testicles had not well been investigated.Here we identified the transcription factor PAX7 is different expressed in spermatogonia in goat dairy. So, we design the experiment to find the function of Pax7, how it can affect the process of self-renewal and proliferation in dairy goat SSCs.In the study, we analyzed the sequencing data of CD49f-positive and negative testicular cells in dairy goat. We also found that Pax7 can regulate the function of SSCs self-renewal.1. The transcripts expression profiling of dairy goat testicular cellsUsing MACS(Magnetic Activated Cell Sorting) technology to purified CD49f-positive and negative cells. We got the transcripts sequencing data by by Hiseq2000 high-throughput sequencing technology. Then we analysis the differentially expressed transcripts data. The results showed that, among the identified novel and known transcripts, 574 and 1426 genes were upregulated with a 2-fold increase in CD49f-positive and negative testicular cells,respectively.2. The function of Pax7 in dairy goat testiclesWe detected the Pax7 expression in dairy goat testis in different age by using the immunohistochemistry method, and we found that the highest expression level of Pax7 in dairy goat testicular tissue was at 3M, following by 6.5M. The whole Pax7 mRNA sequence of dairy goat was obtained by In silicon cloning, and the recombinant vector(pPax7-mCherry-N1) was constructed. We transfected the Pax7 over-expression vector in SSCs and then detected the expression of genes that related with SSCs’ self-renewal and differentiation by QRT-PCR and Immunofluorescence methods. We found that Pax7 could maintain the expression of GFRa1、Plzf 、ID4、 OCT4. All the results varied that the role of Pax7 on mGSCs’ self-renewal was verified.