[Construction of eukaryotic expression vector of glucose-regulated protein 75 gene deletion mutant and its expression in PC12 cells.].
Glucose-regulated protein 75 (Grp75) binds to p53 and inhibits its nuclear translocation, and thus plays a role in cell protection. To investigate whether the binding of Grp75 and p53 would influence the viability of cells, we constructed the eukaryotic expression vector of Grp75 deletion mutant. The deletion mutant gene was obtained by SOE-PCR (gene splicing by overlap extension) and then linked to the pcDNA3.0 vector. The constructed specific expression vector, pcDNA3.0/Grp75(Δ253-282), was identified by restriction enzymes and sequencing. Then we used liposome to transfect the specific vector into PC12 cells. The stable cell strain PC12/Grp75(Δ253-282)(+) was selected by G418 (1 mg/mL). Semi-quantitative RT-PCR and Western blot showed that Grp75 mRNA and protein expressions in PC12/Grp75(Δ253-282)(+) cells were higher than those in PC12 cells. The viability of cells undergoing 0 h, 3 h, 9 h, 18 h and 36 h of glucose deprivation respectively was measured by MTT assay. The results showed that the cell viability of PC12/Grp75(+) group was significantly higher than that of the other two groups, and the cell viability of PC12/Grp75(Δ253-282)(+) group was significantly higher than that of the PC12 group (P<0.05). Hoechst33324 staining was employed to detect cell apoptosis and the results were consistent with the MTT assay results. Western blot results indicated that the expression of p53 in PC12/Grp75(+) cells was lower than those in the other two groups, which might be due to the overexpression of Grp75. These results suggest that the protective role of Grp75 is partly associated with its binding to p53. The above results suggest that Grp75 deletion mutation could to some extent reduce the viability of cells.
Guo
Wei-Wei
WW
Department of Cellular and Genetic Medicine, Shanghai Medical College, Fudan University, Shanghai 200032, China.
Yang
Ling
L
Liu
Xiao-Yu
XY
Liu
Wen
W
Zuo
Ji
J
D000818:Animals; D017353:Gene Deletion; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D014162:Transfection
D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D050505:Mutant Proteins; C018551:glucose-regulated proteins