title: [Packaging of lentivirus carrying gene hβc and overexpression of gene hβc in NB4 cells].
abstract: This study was aimed to overexpress gene hβc in NB4 cells via the method of lentivirus-mediated gene transfer, to observe the differentiation behaviour change of hβc over-expressing NB4 cells treated with IL-3 or GM-CSF, to explore the relationship between hβc gene and the differentiation behaviour of NB4 cells. The targeted hβc gene was amplified by PCR from the cloned vector carrying ORF of hβc. The PCR product containing PmeI and BstBI site introduced by primer was digested, and then cloned into lentivirus vector pRRLSIN.cPPT.PGK/IRES/GFP.WPRE to construct a lentiviral vector carrying hβc, named pLV-hβc. And the pLV-hβc plasmid was confirmed by restriction and sequencing. The recombinant lentivirus was produced by co-transfecting three plasmids into 293T packing cells. After transfection, the lentiviral supernatant was collected to transfect NB4 cells. GFP expression was examined by fluorescent microscope and the expression of hβc gene was detected by Western blot. Then, the NB4 cells over-expressing hβc were treated with IL-3 (10 ng/ml), GM-CSF (10 ng/ml), ATRA (1 µmol/L) respectively, and the CD11b expression, morphology and differentiation behaviour changes of every groups were observed by flow cytometry and microscopy, while NB4 cells transfected with blank lentivirus (NB4-blank cells) were used as controls. The results showed that the recombinant lentivirus vector carrying hβc gene could efficiently transfect NB4 cells and made NB4 cells to stably over-express hβc gene. The expression of CD11b was up-regulated in NB4-hβc cells treated with of IL-3 or GM-CSF, but it was not as obvious as the effect of ATRA, and no morphological change was observed in NB4 hβc cells treated with the IL-3 or GM-CSF. It is concluded that IL-3 or GM-CSF can induce NB4 cells over-expressing hβc to differentiate to neutrophils, but can not make them fully matured.
authors: Fujian Institute of Hematology, Fujian Key Laboratory of Hematology, Department of Hematology, Fujian Medical University Union Hospital, Fuzhou 350001, Fujian Province, China.
authors: Jing-Hui
authors: JH
authors: Yang
authors: Yong
authors: Y
authors: Wu
authors: You-Mei
authors: YM
authors: Zi
authors: Xian-Fang
authors: XF
authors: Li
authors: Xiao-Ying
authors: XY
authors: Liao
authors: Yuan-Zhong
authors: YZ
authors: Chen
mesh_terms: D002454:Cell Differentiation; D002460:Cell Line; D053648:Cytokine Receptor Common beta Subunit / Q000235:genetics*; D005434:Flow Cytometry; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis; D006801:Humans; D007377:Interleukin-3 / Q000096:biosynthesis; D016086:Lentivirus / Q000235:genetics*; D010957:Plasmids; D014162:Transfection
chemical_list: D053648:Cytokine Receptor Common beta Subunit; D007377:Interleukin-3; D016178:Granulocyte-Macrophage Colony-Stimulating Factor