ABSTRACT
We studied the function of connexin 43 (Cx43) gene in Xiao Meishan swine bone marrow mesenchymal stem cells (BMSCs) resulting from overexpression. Cx43 eukaryotic expression vector (pEGFP-Cx43) was constructed and transfected into BMSCs by nucleofector, after detecting the transfection efficiency; the expression of Cx43 was verified by RT-PCR, immunofluorescence and western blotting. Furthermore, we detected its cell cycle and apoptosis through flow cytometry. Our results show that pEGFP-Cx43 plasmid was successfully constructed, and green fluorescence in pEGFP-Cx43 transfected BMSCs was highly expressed with 60% transfection efficiency. In transgenic Xiao Meishan swines BMSCs, the expression level of Cx43 mRNA and protein were up-regulated. Meanwhile, the ability of cell proliferation was significantly increased, and the apoptosis rate was significantly reduced. Taken together, Cx43 overexpression could promote the proliferation of Xiao Meishan swine's BMSCs and markedly reduce their apoptosis, which provides evidence for in vivo research.
Subject(s)
Animals , Animals, Genetically Modified , Apoptosis , Cell Proliferation , Connexin 43 , Metabolism , Flow Cytometry , Genetic Vectors , Mesenchymal Stem Cells , Metabolism , Plasmids , Swine , Genetics , TransfectionABSTRACT
To monitor the trans-differentiation from adult stem cells to germ cells, we analyzed the vasa expression of goat testicular tissues in different ages and constructed the germ cell specific reporting vector pVASA-EGFP. The expression of vasa was verified by RT-PCR and immunofluorescence. The vector pVASA-EGFP was constructed by molecular technology, then transfected into goat bone mesenchymal stem cells (BMSCs) by Lipofectamine 2000. Moreover, we observed the expression of the vector through green fluorescent protein (GFP). Immunofluorescence results show that Vasa was expressed in all groups of goat testicular tissues, RT-PCR results show that the levels of vasa mRNA in 3-month group and 10-month group were significantly higher than that in 10-day group. Sequencing and restriction enzyme results show that the vector was successfully constructed. After transfection and RA treatment, GFP expression was observed, which proved the validity of our reporting system. All the results proved that vasa was expressed in different ages in goat testicular tissues, and the vector pVASA-EGFP is efficient in monitoring the trans-differentiation in vitro, which paves the way for further characterization and screening of the trans-differentiation of goat BMSCs.