[Construction of lentiviral vectors encoding GDNF and GDNF expression in human neural stem cells].
We transfected human neural stem cells using lentiviral vectors encoding glial cell line derived neurotrophic factor (GDNF) to study its expression level in vitro and to get a stable cell line expressing GDNF. First, GDNF gene was sub-cloned into the lentiviral transfer vectors. Then, the recombinant lentiviral supernatants were packaged by 293T cells through three plasmids transient co-transfection method using standard lipofectamine reagent. The viral titers were tested by the transfection efficiency of 293T cells. At the same time, human neural stem cells (hNSC) were transfected under different multiplicity of infection. GDNF gene expression level and protein secretion level of hNSC were tested by real-time PCR and ELISA methods after transfection. Lentiviral vectors encoding GDNF were constructed. Using lentiviral vectors encoding GDNF we successfully transfected human neural stem cells, and got a stable neural stem cell lines over-expressing GDNF. Furthermore, the results indicated that GDNF expression was influenced by the multiplicity of infection. Human neural stem cells could over-express GDNF through lentivial vectors tranfection. Its gene expression level and protein expression level correlate with the multiplicity of infection.
Wang
Shuyan
S
Cell Therapy Center, Xuanwu Hospital, Capital Medical University, Beijing 100053, China.
Ren
Ping
P
Xie
Shu
S
Zhu
Wanwan
W
Wang
Yang
Y
Guan
Yunqian
Y
Zhang
Yu
Y
D053595:Embryonic Stem Cells / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D009417:Nerve Tissue / Q000166:cytology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D051100:Glial Cell Line-Derived Neurotrophic Factor; D011994:Recombinant Proteins