Gene Cloning And Inducing Expression Of β-NGF And Effect On Limbal Stem Cells

Objective: To construct recombinant lentiviral vector carrying rat β-NGF gene,which can be used by inducing expression of the Tet-on 3G tetracycline induced expression system,and then to observe its expression in Limbal stem cells.Methods: Total RNA was extracted from rat brain as the template. By gene recombination technique,rat β-NGF gene was inserted into lentiviral vector as pLVX-TRE3G-IRES-β-NGF. We collected the supernatant of virus including the recombinant vector of pLVX-TRE3G-IRES-β-NGF and pLVX-Tet3 G from packaging cells, respectively, and then infected blank HEK293 FT cells. The expression of β-NGF was induced with different doses of doxycycline(Dox)(divided into non-transfection group, 0, 100, 500 and 1000 ng/ml of Dox inducing group). 48 h later, cells were collected and analyzed by western blot assay. At the same time,the supernatant was detected by ELISA assay for the secretion and used as conditional medium for PC-12 cells. After primary cultured, LSCs were allocated to four groups with different doses of doxycycline(Dox)(divided into control group( non-transfection group), 1000, 100 and 1000 ng/ml of Dox inducing groupA、B、C), observed at 48 h. Expression of p63 was examined with Immunofluoresence staining.Results: A band of β-NGF gene was about 726 bp. After transfection, β-NGF protein was detected to express on 293 FT cells and supernatant following the increase of Dox dose. The neuron-specific enolase(NSE) was expressed on PC-12 cells after induced by conditional media. Then, β-NGF protein was too detected to express on LSCs following the increase of Dox dose; The experimental group about expressing quantity of NGF, p63, TrkA, p38 difference were statistically significant.Conclusion: We show here that theβ-NGF gene could be expressed successfully on HEK 293 FT cells by Tet-on 3G induced expression system, and the protein has effected on inducing PC-12 cells differentiation into neuron-like cells. We also show that the β-NGF gene could be expressed successfully on Limbal stem cells by Tet-on 3G induced expression system, and the protein maintains their stemness.