Stably expression human α-synuclein gene in PC12 cells by lentivirus and apoptosis detection

Abstract

Objective: Construction and identification of wild-type and mutant human α-synuclein (SNCA) gene lentiviral expression vector, and its stable transfection into the rat pheochromocytoma cells. Methods: The genes were synthesized with particular primer, amplified by PCR and cloned into the lentiviral vector expression plasmid pGC-FU (with green fluorescent protein(GFP) gene) to construct a lentiviral vector expression plasmid pGC-FU-SNCA-GFP and pGC-FU-SNCA-GFP. After digestion and sequencing, pGC-FU-SNCA-GFP, pGC-FU-SNCA-GFP plasmid and packaging plasmid pHelper1.0, pHelper2.0 were co-transfected into rat pheochromocytoma cells (PC12 cells). A stable transfection was established in the PC12 cells. Results: By detecting the level of tagged protein of GFP and the target protein, the pGC-FU-SNCA-GFP and pGC-FU-SNCA-GFP expression in target cells was verified. MTT assay was employed to detect cell apoptosis in lentiviral pGC-FU-SNCA-GFP transfected group, lentiviral pGC-FU-SNCAmu-GFP transfected group (experimental groups), without virus group (control group) and empty vector group (total four groups cells). After transfection, at different timepoints (1, 2, 3, 4 and 5 d) the proliferation of cells was slowed (the F value was 4.534, 196.285, 411.829, 1282.049, 3135.559, all P < 0.05). PI single staining was used to examine the cell cycle. The percentages of G1 phase, G2 phase, M phase cells were all statistically significant (the F value was 885.79, 45.03, 207.11, all P < 0.05). The percentage of G1 phase cells in the four groups cells increased significantly (CON group: 59.10 ± 0.35, NC group: 68.24 ± 0.60, OE group: 71.73 ± 0.11, OE group: 74.66 ± 0.35). Conclusion: This study constructs a foundation for further investigation on the basic function of SNCA and apoptosis related diseases.