The study of the cotransfection of pcDNA3.1his-hTH and pEGFP-C2 in the neural stem cells derived from bone marrow stromal cells

Objective To construct a recombinant plasmid pcDNA3.1his-hTH which can express hTH (human tyrosine hydroxylase) gene and 6-his in eukaryocyte and cotransfect neural stem cells derived from bone marrow stromal cells (NSCs-BMSCs) with pEGFP-C2, then observe the expression of hTH gene in NSCs-BMSCs. Methods With the technology of gene re-arrangement, hTH gene in pWAV2-TH plasmid was subcloned into pcDNA3.1his vector to obtain pcDNA3.1his-hTH plasmid, then its correctness was evaluated by the means of restriction enzyme analysis and sequencing. pcDNA3.1his-hTH and pEGFP-C2 was contransfected into NSCs-BMSCs by NucleofectorTM. After 24h, the transient expression of EGFP was observed under fluorescence microscope,and the antigen expression of TH and 6-his were measured by immunohistochemistry.Meanwhile, the mRNA of TH gene was detected by RT-PCR. Results ⑴ Correct construction of pcDNA3.1his-hTH was identified by methods of restriction enzyme analysis and nucleotide sequence determination.⑵ Green fluorescence was emitted from transfection cell under fluorescent microscope after 24h, then immunohistochemistry showed large parts of transfection cells which expressed TH and 6-his and displayed positive staining, and the expression of hTH gene was detected by RT-PCR after 10d . Conclusions pcDNA3.1his-hTH, which was successfully constructed, and pEGFP-C2 were cotransfected in NSCs-BM of Rhesus monkey. The system could be as a technic platform to detect transfection efficiency in vitro and track grafted cells to treat PD in vivo, because hTH、EGFP and 6His genes were able to be expressed in NSCs-BMSCs.