[Construction of rat bdnf gene lentiviral vector and its expression in mesenchymal stem cells].
Recently, mesenchymal stem cells (MSCs) have been one of the target cells of gene engineering. To construct the lentiviral (LV) vectors carrying the brain-derived neurotrophic factor (Bdnf) gene, the rat mesenchymal stem cells (rMSCs) were infected and finally the Bdnf gene-modified rMSCs was obtained. The CDS region of the rat Bdnf gene was obtained with reverse transcriptase-polymerase chain reaction (RT-PCR), and the transfer plasmid (PNL-BDNF-IRES2-EGFP) of the LV vector was constructed. The three plasmids of LV vector: PNL-BDNF-IRES2-EGFP, HELPER, and VSVG were cotransfected to 293T cells to produce the LV vectors, which enabled the coexpression of the Bdnf gene and the enhanced green fluorescent protein (Egfp) gene. rMSCs were separated from the bone marrow of 2-month-old F344 rats, cultured in vitro, and identified. rMSCs were infected by the LV vectors that were produced already and were identified with fluorescent microscope, RT-PCR, immunocytochemical staining, and western blot. The result of sequencing showed that the sequence of the cloned Bdnf gene was consistent with that reported in the GenBank. The PNL-BDNF-IRES2-EGFP plasmid that was identified showed the correct sequence. After the 3 plasmids of LV vectors were cotransfected to the 293T cells, considerable green fluorescence in 293T cells was observed under the fluorescent microscope; the supernatant was collected and concentrated using ultracentrifugation, and the titer of the replication-defective LV vector particles measured was found to be 6.7 x 10(7) TU/mL. After the constructed LV vectors infected the rMSCs, the results obtained using RT-PCR, immunocytochemical staining, and western blot showed that the expression of BDNF in the Bdnf-rMSCs group (experimental group, EG) was significantly higher than that in the PNL-IRES2-EGFP-rMSCs group (mock group, MG) and the rMSCs group (control group, CG) at both mRNA and protein levels. LV vectors carrying the Bdnf gene were constructed successfully. The Bdnf gene-modified rMSCs could express BDNF to a higher degree. This greatly facilitates the next step in the study, such as the long period of therapeutic observation of cerebral ischemia with Bdnf gene-modified rMSCs.
Huang
Dong-Yu
DY
Department of Neurology, The First Hospital Affiliated to Fujian Medical University, Fuzhou 350005, China.
Zhang
Zhi-Jian
ZJ
Chen
Bai-Ling
BL
Wu
Xiu-Li
XL
Wang
Ning
N
Zhang
Yan-Ding
YD
D000818:Animals; D015153:Blotting, Western; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D016086:Lentivirus / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D051381:Rats; D011916:Rats, Inbred F344; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic
D019208:Brain-Derived Neurotrophic Factor; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins