Can Be Induced Testis-specific Promoter Cre Tools Mice To Establish And Hrb2 Cell Function

Objective: To construct Sertoli cells and spermatogonia-specific Cre tool miceMethods: DNA fragments AMH and STRA8 were amplified by PCR and cut by EcoR I and Spe I, the products were ligated to a digested vector PCAG-ERT2-CRE-ERT2 using the same restriction endonucleases. Then plasmid AMH-ERT2-CRE-ERT2 and STRA8-ERT2-CRE-ERT2 were digested by Spe I to get linear DNA fragments. The DNA fragments were diluted into 2ng/μl and directly injected into the nucleus of C57BL / 6 oospores. After in vitro culturing of 1-2 hours, the eggs were transplanted into the pseudopregnant ICR female mice's oviducts. After pregnancy, DNAs were extracted from filial mice tails, and checked the positive transgenic mice by PCR.Results: Successfully constructed AMH-ERT2-CRE-ERT2 and STRA8-ERT2-CRE-ERT2 plasmid. DNAs from AMH injected a total number of 205 eggs and 180 viable fertilized eggs were transplanted into the oviducts of 9 ICR pseudopregnant female mice, and 94 offspring were obtained. PCR analysis showed that 9 transgenic mice were positive. And DNAs from STRA8 injected a total number of 164 eggs and 147 viable fertilized eggs were transplanted into the oviducts of 7 ICR pseudopregnant female mice, and 58 offspring were obtained. PCR analysis showed that 8 transgenic mice were positive.Conclusion: By prokaryotic injection, Sertoli cells and spermatogonia-specific inducible Cre recombinase transgenic tool mouse C57BL/6 FVB-Tg(AMH-ERT2-CRE-ERT2) and C57BL/6 FVB-Tg(STRA8-ERT2-CRE-ERT2) were successfully prepared. Purpose: HRB2(HIV-1 rev binding protein 2)is a protein constructed by 381 amino acid residues.The HRB2 gene has an ORF of 1143bp. Lots of eukaryotic organisms express HRB2. Studys on them find that, HRB2s in these organisms are homology, and they play important roles in a great part of vital movement, such as adjustment of ribosomal synthesis, production of protein-protein interactions and subcellular localization. HRB2 in Saccharomyces cerevisiae has bene researched deeply, but not in human. In our study, we analyzed HRB2 from human in protein constitution, homology, subcellular localization, expression in cell lines, RNA interference, the effect on cell cycling and its protein interaction with NYD-SP14.Methods: By use of software, such as NCBI/SMART/Unigene and clustalx, we suppose the homology, subcellular localization and Biological function of HRB2. RT-PCR is used to find HRB2's expression in cell lines. And also we use fluorescence microscope to find HRB2's subcellular localization after construction of plasmids expressing GFP-HRB2 fusion proteins. We construct RNAi plasmids and use them to degrade the expressment of HRB2 in cell line called 293T. Then the effects of HRB2 on cell cycling were detected by scratch-healing assays ,the growth curve experiments and flow cytometry analysis. Finally, by the use of co- localization, we find that, the protein-protein interaction is existence between HRB2 and NYD-SP14.Results: HRB2 is expressed in lots of eukaryotic organisms, and it is conservative in these organisms. HRB2 protein contains a KH domain, which is widely exist in eukaryotic organisms and plays important roles in lifes. By the use of RT-PCR, we found that HRB2 is expressed in cell lines as Hela,293T and HEK TER. Studys on cell lines find that, HRB2 from human is localized in nucleolus in cells. We designed four plasmids which contains RNA interference sequence for HRB2. By the use of Real-Time PCR, we found that, the interference sequence for HRB2 we used can degrade HRB2's expression, and No.3 and No.4 degraded mostly. The results of scratch-healing experiments and growth curve indicated that the growth of cell with the No.3 and No.4 was inhibited. However, the results of flow cytometry analysis showed no significant difference. Endogenous NYD-SP14 is localized in cytoplasm in cells. While in the cells(Hela) in which HRB2 is over expressed, we found that NYD-SP14 is localized in nucleolus mostly by immunofluorescence analysis. HRB2 and NYD-SP14 bind to each other, and their complex is localized in nucleolus in vivo.Conclution: HRB2 is expressed in lots of eukaryotic organisms, and it is conservative in these organisms. HRB2 is localized in nucleolus in cell lines. We screened an ideal interference sequence for HRB2, and found HRB2 gene had an impact on the cell cycling. In addition the interaction between HRB2 and NYD-SP14. Their complex is localized in nucleolus in vivo.