山羊生殖细胞特异报告载体pVASA-EGFP的构建及表达

Construction of goat germ cell specific reporting system pVASA-EGFP

摘要:

为监测成体干细胞向生殖细胞分化的过程，分析生殖细胞特异性DEAD-box家族ATP依赖RNA解旋酶vasa在不同日龄山羊睾丸组织中的表达情况并构建山羊生殖细胞特异性报告载体pVASA-EGFP。通过免疫荧光及RT-PCR法监测vasa的表达情况，利用分子技术构建报告载体pVASA-EGFP，脂质体法转染山羊骨髓间充质干细胞 (Bone mesenchymal stem cells，BMSCs)，经过视黄酸 (Retinoic acid，RA) 诱导后，观察绿色荧光蛋白表达情况以鉴定该报告载体的有效性。免疫荧光结果显示，Vasa在性成熟不同阶段的山羊睾丸组织中均有表达，RT-PCR结果表明vasa基因在3月龄、10月龄山羊睾丸组织中显著高于10日龄组。测序及酶切鉴定结果表明，扩增的vasa基因启动子片段成功连至N1载体，转染BMSCs后经4 d的RA诱导，发现有绿色荧光蛋白表达，表明成功构建了山羊vasa基因启动子调控的报告载体pVASA-EGFP。以上结果表明，vasa基因在不同日龄山羊睾丸组织中均有表达，所构建的山羊生殖细胞特异性报告载体pVASA-EGFP具有示踪山羊成体干细胞向生殖细胞分化过程的能力，为下一步监测山羊BMSCs向生殖细胞分化的过程提供了鉴定和筛选方法。

Abstract:

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.