Abstract:

Objective To build a cell line stably expressing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid(AMPA) receptor GluA1 subunit. Methods The GluA1 gene was amplified by PCR and loaded into the lentiviral expression vector PLV.0-green fluorescent protein(GFP). HEK293T cells were transfected by using the four plasmid system. Puromycin was used for screening stable cell lines. The monoclonal cells were identified by Real-time PCR, Western blotting, immunofluorescence and whole cell patch clamp. Results The amplified product identified by colony PCR was consistent with the molecular weight of GluA1 (2724 bp). Further sequencing result showed that the sequence inserted into the lentiviral vector was consistent with the GluA1 sequence; HEK293T stable cell lines screened by puromycin selection were confirmed by Real-time PCR, Western blotting and immunofluorescence. GluA1 expression was correct in the experimental group, but not expressed in the control group. Immunofluorescent staining showed that the expression of GluA1 protein was relatively uniform on each cell surface. Stable cell lines were detected in voltage clamp mode with 10 mmol/L glutamate in extracellular fluid, and no electrical signal was detected in the empty vector group, whereas 5-40 pA electrical signal was recorded in the GluA1 overexpressing group. Conclusion We successfully established a HEK293T monoclonal stable cell strain expressing GluA1 subunit.

Key words: GluA1 subunit, Lentiviral, Monoclonal, Cell strain, Real-time PCR, Western blotting