[Experimental study of rat mesenchymal stem cells transfected with Sonic Hedgehog gene].
ABSTRACT To evaluate the feasibility of genetic modification of mesenchymal stem cells (MSC) with Sonic Hedgehog (Shh) gene.
The pcDNA3.1-Shh eukaryotic expression plasmid was constructed and its correctness evaluated by the restriction enzyme analysis and sequencing. MSC were isolated from Wistar rats by density gradient centrifugation and purified, transfected with pcDNA3.1-Shh, blank plasmid pcDNA3.1(-) or pmaxGFP respectively by Nucleofector(TM). The protein expression of Shh in MSC was detected by Western blot after 48 hours.
Correct construction of pcDNA3.1-Shh was identified by the methods of restriction enzyme analysis and nucleotide sequence determination. The expression of green fluorescent protein (GFP) could be observed by fluorescence microscopy after 48 hours. The expression of Shh gene was detected by Western blot. But the MSC transfected with empty plasmid expression was not detected.
Recombinant Eukaryotic expression plasmid pcDNA3.1-Shh is successfully detected in rat MSC. It may provide experimental rationales for the future gene therapy.