TRKC基因体外转染大鼠神经干细胞的方法

目的探讨体外转染TRKC基因的神经干细胞的制备方法。方法利用自行合成的引物进行PCR反应,定向克隆构建TRKC-cDNA质粒并进行DNA测序分析,将pEGFP-C1质粒和TRKC-cDNA质粒进行酶切、重组,构建出pEGFP-C1-TRKC质粒。用脂质体将重组质粒转染到体外培养的大鼠神经干细胞中。用W estern b lot、免疫荧光和MTT法观察转染基因的表达和转基因后神经干细胞的增殖活性。结果成功构建了TRKC-cDNA质粒,DNA测序证明所获得的目的基因与公布序列的一致性为99%,所获得的重组子符合研究要求。pEGFP-C1-TRKC质粒转染到神经干细胞后,宿主细胞能高效、稳定地表达目的基因产物GFP和TRKC。在NT-3作用下转TRKC基因神经干细胞的增殖活性有明显增强。结论以质粒为载体、脂质体为介导可成功地把TRKC基因转染到神经干细胞内,转染基因表达好且具有功能。

Method of transferring TRKC gene into rat neural stem cells in vitro

Objective To creat a method for the preparation of neural stem cells(NSCs) with over-expressing TRKC gene.Methods First,the primer of TRKC was synthesized,TRKC gene was produced with PCR,then TRKC-cDNA expressed plasmid was constructed with directional cloning,and DNA sequencing analysis aws performed.After that,(pEGFP-C1-TRKC) plasmid was constructed with enzyme digestion and recombination of(pEGFP-C1) and TRKC-cDNA plasmids and verified.Next,pEGFP-C1-TRKC plasmid was transferred into rat NSCs with lipofectamine.Then TRKC gene expression in NSCs was observed by Western blot,immunofluorescent staining and MTT.Results The TRKC-cDNA expressed plasmid was successfully constructed,and DNA sequencing analysis proved that the target gene has good consistency(99%) with the reported TRKC sequence.The constructed(pEGFP-C1-TRKC) plasmid meat the demand of this experiment,verified by enzyme digestion and electrophoresis.After the pEGFP-C1-TRKC plasmid was transferred into NSCs,NSCs efficiently and stably expressed the products of the target genes(GFP and TRKC).MTT showed apparent improvement of the proliferation viability of the transferred cells with the existence of NT-3. Conclusion The methods can successfully transfer TRKC gene into NSCs.The transferred gene can over express products on NSCs and the products functions well.