Construction Of The Interference Vector Of Mll1and Its Effect On The Spermatogenesis Of Mouse

Spermatogonial stem cells (SSCs) are the only stem cells in adult male germline,which can transmit genetic information to the next generation, have self-renewalpotential in the seminiferous tubule, and continuously support spermatogenesis for thelife time in male animals. While spermatogenesis as an efficient system is controlledby a series of transcriptional regulation and chromatin modification, the histonemethyltransferases involve in histone methylation to change the chromatinmodification and play an important role in many biological processes of mammals,such as heterochromatin formation, X chromosome inactivation and transcriptionalregulation. In this study we examined the mRNA expression level ofmethyltransferase MLL1(Mixed-lineage leukaemia1) in different tissues; thenconstructed a shRNA interference vector and packaged its lentivirus; tested its effectsboth in vivo and in vitro of mouse spermatogonial proliferation, as well as theirimpacts on the regulator genes of spermatogonia. The main findings are as follows:1. According to the data based on GenBank, we designed and synthesizedprimers of Mll1. Its expression level in mouse heart, liver, spleen, lung, kidney, testis,ovary, and GC-1spg cell lines showed that the lung expressed the highest, followedby the spleen and ovary, testis got a higher expression, while the heart, liver and GC-1spg also showed the expression.2. Using online shRNA design software, we confirmed Mll1interference andcontrol shRNA sequences. These two sequences were cloned successfully to theCD513B-U6vector respectively. Then we used the third generation of lentiviralpackaging system to obtain the interference lentivirus of which the viral titer was1.5×107TU/mL.3. We transduced Mll1interference and control lentivirus to the GC-1spg cell lines. After puromycin selection, the expression level of Mll1in GC-1spg cells stablydecreased with a60%down regulation compared to the NC-lenti and mock groups.The proliferation curve of cell count experiments showed that Mll1barely had effectson the proliferation of mouse spermatogonia.4. We injected the Mll1interference lentivirus and NC lentivirus directly into theseminiferous tubules of unilateral testis of six-week-old Kunming white mice in vitro.The percentages of tubules with spermatogenesis had a significant decrease betweenMll1-lenti-RNAi and NC-lenti group.5. Finally, the detection of spermatogonial proliferation, differentiation andself-renewal related genes displayed that the transcription factor ETV5associatedwith self-renewal was down-regulated by72%after Mll1interfering.