[Construction of a lentiviral vector carrying human bcl-2 gene and its expression in human ovarian granulosa cells].
OBJECTIVE
To construct a lentiviral vector carrying human bcl-2 gene and investigate its expression in human ovarian granulosa cells (GCs).


METHODS
Human bcl-2 gene was amplified from the plasmid pCMV-SPORT6 using PCR and subcloned into the lentiviral vector pGC-FU to construct the lentiviral expression vector pGC-FU- bcl-2. The bcl-2 gene insert was confirmed by restriction enzyme digestion and sequencing. The recombinant lentiviruses generated by 293T cells co-transfected with pGC-FU-bcl-2 and the packaging plasmids pHelper1.0 and pHelper2.0. The resulting recombinant lentivirus GC-FU-bcl-2 carrying bcl-2 and EGFP genes were then used to infect human ovarian granulosa cells. EGFP and bcl-2 protein expressions in 293T and human ovarian GCs were detected by fluorescent microscope and Western blotting.


RESULTS
The plasmid pGC-FU-bcl-2 carrying the correct bcl-2 gene sequence could be expressed in human ovarian GC cells, and the recombinant lentivirus GC-FU-bcl-2 was generated by the packaging 293T cells. Stable expression of EGFP and bcl-2 proteins were detected by fluorescent microscope and Western blotting in 293T and human ovarian GCs after the infection. The recombinant lentivirus efficiently delivered bcl-2 gene into human ovarian GCs, in which bcl-2 expression was expressed efficiently and stably.


CONCLUSIONS
The recombinant lentivirus GC-FU-bcl-2 has been successfully constructed, which is capable of delivering the target gene bcl-2 into human ovarian GCs for its stable expression.
Wang
Xue-feng
XF
Department of Obstetrics and Gynecology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China. douwangxuefeng@163.com
He
Yuan-li
YL
D002478:Cells, Cultured; D005260:Female; D019254:Genes, bcl-2 / Q000235:genetics*; D005822:Genetic Vectors; D006107:Granulosa Cells / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D010053:Ovary / Q000166:cytology / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins