In vitro non-viral lipofectamine delivery of the gene for glial cell line-derived neurotrophic factor to human umbilical cord blood CD34+ cells.
Using a lipofection technique, we explored a non-viral delivery of plasmid DNA encoding a rat pGDNF (glial cell line-derived neurotrophic factor) to CD34+ cells derived from human umbilical cord blood (HUCB) cells in order to obtain cells stably expressing the GDNF gene. The target gene GDNF was amplified from cortex cells of newborn Sprague-Dawley rats by reverse transcriptase polymerase chain reaction (RT-PCR) and inserted into vector pEGFP-N1 to construct the eukaryotic expression vector pEGFP/GDNF. The positive clones were identified by sequencing and endonuclease digestion. The expression of pEGFP/GDNF-transfected HUCB cells CD34+ was examined by ELISA. Single fragment of 640 bp was obtained after the rat GDNF cDNA was amplified by RT-PCR. Two fragments of about 4.3 kb and 640 pb were obtained after digestion of recombinant plasmid pEGFP/GDNF with XhoI/KpnI. The nucleic acid fragment of 640 bp was confirmed to agree well with the sequence of GDNF gene published by GenBank. The expression of GDNF mRNA and the level of GDNF from pEGFP/GDNF-transfected CD34+ cells were increased substantially, compared with pEGFP control plasmid transfected CD34+ cells (P<0.05). Moreover, co-culture of primary rat cells with the pEGFP/GDNF-transfected CD34+ cells promoted enhanced neuroprotection against oxygen-glucose deprivation induced cell dysfunctions. The present results support the use of the non-viral plasmid liposome for therapeutic gene expression for stem cell therapy.
Yu
Guolong
G
Department of Cardiology, Xiangya Hospital, Southern Central University, Changsha, China. yuguolong123@yahoo.com.cn
Borlongan
Cesar V
CV
Ou
Yali
Y
Stahl
Christine E
CE
Yu
SeongJin
S
Bae
EungKyung
E
Kaneko
Yuji
Y
Yang
Tianlun
T
Yuan
Chunjun
C
Fang
Li
L
D000818:Animals; D000831:Animals, Newborn; D018952:Antigens, CD34 / Q000032:analysis*; D016923:Cell Death / Q000235:genetics / Q000502:physiology; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology; D049109:Cell Proliferation; D002540:Cerebral Cortex / Q000378:metabolism; D018920:Coculture Techniques; D005312:Fetal Blood / Q000166:cytology*; D018014:Gene Transfer Techniques; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000235:genetics* / Q000378:metabolism; D005947:Glucose / Q000172:deficiency; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008055:Lipids; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D018952:Antigens, CD34; D051100:Glial Cell Line-Derived Neurotrophic Factor; D008055:Lipids; C086724:Lipofectamine; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005947:Glucose