[Establishment of a stable transfectant 32D/Gfi1 cell line by recombinant lentiviral expression vector].
The aim of study was to establish the packaging system of the recombinant lentiviral vector encoding Gfi1 gene for eukaryotic expression and to realize the efficient, stable expression of Gfi1 32D cells so as to provide effective platform for further studying the development of Gfi1 gene in hematologic malignancies. The three-plasmid recombinant lentiviral vector consisting of transfer plasmid (pLOX-Gfi1/pLOX), the packaging plasmid (pCMVDeltaR8.2) and the envelop plasmid (pMD.G) was prepared and purified. Human embryonic kidney 293T cells were cotransfected with the three plasmids by lipofectamine 2000. After transfection for 48 hours, the viral supernatant was collected and the target cell 32D was transfected with the recombinant lentivirus; the Gfi1 integration and expression in 293T and 32D cells were detected by Western-blot. The results showed that the three plasmids of lentivirus could be transfected into 293T with high efficiency and packaged successfully, and the Gfi1 protein could be detected by fluorescent microscopy. The recombinant lentiviruses carrying Gfi1 could transfer and deliver Gfi1 gene to 32D cells, and the Gfi1 expression in 293T and 32D cell could be detected by Western blot. It is concluded that the recombinant lentivirus carrying Gfi1 can deliver target gene to 32D cells with high efficiency, and the expression of Gfi1 protein is stable in 32D.
Huang
Min
M
Department of Hematology, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
Ou
Dong-Mei
DM
Wu
Jiang
J
Zhao
Xia
X
Xu
Jin-Huan
JH
Zhang
Xiao-Mei
XM
Zhang
Yi-Cheng
YC
D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D010957:Plasmids; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection
D004268:DNA-Binding Proteins; C498275:GFI1 protein, human; D014157:Transcription Factors