Construction and identification of human glial cell-derived neurotrophic factor gene-modified Schwann cells from rhesus monkeys.

Abstract

The objective of this study was to construct stable rhesus monkey Schwann cells (SCs) modified with the human glial cell-derived neurotrophic factor (hGDNF) gene. hGDNF gene amplification was performed with pUC19-hGDNF as templates, and then the coding sequence of hGDNF was inserted into the eukaryotic expression vector pBABE-puro to obtain the recombinant vector pBABE-puro-hGDNF. The recombinant vector pBABE-puro-hGDNF was identified with restriction enzyme, and then underwent DNA sequencing. SCs from rhesus monkeys were transfected with the recombinant vector pBABE-puro-hGDNF, and then the expression levels of mRNA and protein of the hGDNF gene were determined with real-time fluorescence quantitative PCR and Western blot, respectively, in the transfected SCs. The biological activity of GDNF gene-modified SCs (GDNF-SCs) was assessed by MTT assay. The length of the hGDNF coding sequence of PCR products was 569 bp. After the recombinant eukaryotic expression vectors were digested with restriction enzyme, there was a specific segment of 596 bp. The results of DNA sequencing of the specific segment of 596 bp were the same as that of hGDNF in GenBank, suggesting that the hGDNF gene was successfully inserted into the recombinant retrovirus vectors. The expression levels of mRNA and protein were significantly higher in transfected SCs as compared to nontransfected SCs (p<0.05). MTT assay indicated that the OD value was significantly higher in GDNF-SCs group than in SCs and DMEM groups (p<0.05). hGDNF-SCs can steadily and efficiently release hGDNF. This study provides a basis for cell therapy of nerve injury.