Construction of human breast cancer cell lines HS578T and MDA-MB-231 overexpressing GRP78
Objectives To construct human breast cancer cell lines HS578T and MDA-MB-231 that overexpress the endoplasmic reticulum stress marker glucose-regulated protein 78(GRP78),and to provide a basis for exploring the mech-anism of GRP78 in the development and progression of breast cancer.Methods The lentiviral vector pCDH-CMV-GRP78-HA-EF1-Puro plasmid expressing GRP78 was constructed by molecular cloning technology,and the plasmid was amplified by PCR method.Then the plasmid was cut by EcoRⅠ and NotⅠ restriction enzymes,and the digestion products were identified by sequencing.The pCDH-CMV-GRP78-HA-EF1-Puro plasmid was constructed successfully.293FT cell line(clonal isolate)in the logarithmic growth phase was added into a culture dish with pCDH-CMV-GRP78-HA-EF1-Puro mixture,and was cultured for 48 h;the supernate containing GRP78 lentiviral particles was collected and stored for later use.In addition,human breast cancer cell lines HS578T and MDA-MB-231 in the logarithmic growth phase were cultured in the medium containing supernate of GRP78 lentiviral particles.After 48 h culture,1:500 diluted purinycin was added to the medium,and the cell lines of HS578T and MDA-MB-231 with overexpression of GRP78 were screened.The GRP78 of HS578T and MDA-MB-231 cells was detected by Western blotting after being cultured with purinamycin for 48 h.The GRP78 protein in HS578T and MDA-MB-231 cells was localized by confocal laser microscopy.Results The cell lines of HS578T and MDA-MB-231 with overexpression of GRP78 showed obvious GPR78 protein electrophoretic bands around 78 kd.There was GRP78 protein expression in HS578T cells,which was mainly distributed in the cytoplasm.Conclusions We successfully constructed MDA-MB-231 and HS578T cell lines with GRP78 overexpression,and GRP78 was mainly ex-pressed in the cytoplasm.
glucose-regulated protein 78breast carcinomalentiviral vector
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