Study On Cloning And Expression Of Rat Glial Cell Line-derived Neurotrophic Factor

In 1993 GDNF was purified and cloned as one of neurotrophic factors and it can specially promote the survival of midbrain dopaminerigic neurons. After finding GDNF, which is structurally a new distant member of transforming growth factorβ(TGF-β) family, quantities of experiments were carried on mostly concentrated in the upregulations of it to a variety of neurons, and have achieved a lot. At the same time, many studies of GDNF on the neurodegenerative disease are focal spots. Up to now, the transfer of GDNF gene in vivo is a main research approach in all of the experiments to the treatment of PD, ALS and other neurodegenerative disease. PD is characterized by the degeneration or loss of nigrostriatal dopaminergic neurons. It seems that GDNF is the most promising gene for gene therapy in the central nervous system. Therapeutic tools for PD including drug treatment, various kinds surgeries and brain tissue transplantation et al, but there is no method which can prevent or halt the progress of this disorder. Current gene therapy models for PD are described in two parts: genetic transfer of the genes encoding several neurotrophic factors or other antiapoptotic factors (such as bcl-2) for protection of dopaminergic neurons, and genetic transfer of the biosynthetic enzymes for dopamine sythesis(i.e. TH).The studies showed that GDNF was a potent factor for protection of nigral dopaminergic neurons against toxin-induced degeneration in vivo. For this reason, through mainly using molecular cloning, cell culture and expression in animal muscle et al. We cloned the rat GDNF cDNA, then constructed the prokaryotic expression plasmid pET32a-GDNF and the eukaryotic expression plasmid pEGFP-GDNF, to confirm its biological activity of gene transfer through IPTG revulsion and infection protocol ex vivo. We injected pEGFP-GDNF in rats' muscle of posterior limb and initially understanded expression information in vivo, it is the best way of gene therapy. The main methods and results in this study showed as follows:1. Total RNA was extracted from rat brain tissues. According to molecular cloning: a laboratory manual the first and second cDNA chains are synthesized and added PCR primers to amplify the target gene. After amplified with PCR, the section about 636bp is obtained. This section is cloned into carrier pMD-18T so that we got reconstructive pMDI 8-T-GDNF.2. The section was cloned into carrier pGEM-T and we got reconstructive pGEM-T-GDNF, after using restriction endonuclease to shear pGEM-T-GDNF, pET32a, pEGFP-C1, joining them to construct recombinant expression plasmids, the recombinant plasmids are identified by digestion, PCR and confirmed by sequencing.3. The prokaryotic recombinant plasmid was then transfected into E.coli BL21(DE3) for transient expression, the protein was detected by SDS-PAGE and western-blot et al.4. The eukaryotic recombinant plasmid was then transfected into CHO by cationic polymerization for transient expression, the efficiency of gene transfer was detected by RT-PCR, immunocytochemical technique and fluorescence microscope.5. The eukaryotic recombinant plasmid was injected into BABL/c' s muscle of posterior limb, and the expression information was detected by immunohistochemistrical technique and fluorescence microscope.Results:①Cloning ofGDNF geneObtaining cDNA by reverse transcription from total RNA. We acquired 636bp of GDNF target gene, we got a target gene strap and a vector strap after using restriction endonuclease to shear the recombinant plasmids, it showed that the two sequences were completely same comparing between sequenceing and Genebank report.②The prokaryotic expression of GDNF geneTo obtain expression product of GDNF, we successfully constructed the recombinant expression plasmids pET32a-GDNF, the prokaryotic expression protein was detected by SDS-PAGE and western-blot.③The eukaryotic expression of GDNF geneTarget gene had been integrated in cells by cationic polymerization, we can detect the expression of pEGFP-GDNF in CHO cells. We achieved 636bp section by RT-PCR and showed green fluorescence in CHO intracytoplasm. We injected pEGFP-GDNF into rat muscle green fluorescence and immunohistochemical staining were successfully employed to determine the expression of GDNF in muscle. Above results hinted the further expression of EGFP gene and GDNF gene and showed expression successfully.Conclusions:①We had cloned the total sequence of rat GDNF with molecular biological technology and checked the sequence by sequencing, it showed that we obtained the integrity rat GDNF gene fragement correctly.②We have cloned GDNF gene from pGEM-T vector into pET32a, and target gene has been integrated in E.coli B L21(DE3), it showed that pET32a-GDNF gained expression by SDS-PAGE and Western-blot.③GDNF gene had been linked with pEGFP-CI vector, pEGFP-GDNF was transfected into CHO cells.We have successfully detected the expression information by RT-PCR, fluorescence detection and immunohistochemistry. We injected pEGFP-GDNF into rat muscle green fluorescence and immunohistochemical staining were successfully employed to determine the expression of GDNF in muscle. The success of expression laid the foundation for further obtaining GDNF protein of recombination activity and in gene therapy research and its clinical utility in the future for nervous system disease.