Establishment of monoclonal cell strain stably expressing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluA1
Establishment of monoclonal cell strain stably expressing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluA1
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DOI：
10.16098/j.issn.0529-1356.2018.05.005
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发表时间：
2018-10-01
期刊：
影响因子：
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通讯作者：
Guo Guo-qing
中科院分区：
文献类型：
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作者：
Li Jiong;Zhang Ji-feng;Guo Guo-qing
目的建立稳定表达α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）受体GluA 1亚基的细胞系。方法用PCR方法扩增GluA 1基因，并将其装入慢病毒表达载体PLV中。0-绿色荧光蛋白（GFP）。用四质粒系统转染HEK 293 T细胞。嘌呤霉素用于筛选稳定的细胞系。采用Real-time PCR、Western blotting、免疫荧光和全细胞膜片钳技术对单克隆细胞进行鉴定。结果菌落PCR扩增产物与GluA 1的分子量（2724 bp）一致。进一步测序结果显示，慢病毒载体中插入的序列与GluA 1序列一致;经嘌呤霉素筛选的HEK 293 T稳定细胞系经Real-time PCR、Western blotting和免疫荧光检测证实。GluA 1在实验组中表达正确，而在对照组中不表达。免疫荧光染色显示GluA 1蛋白在各细胞表面表达相对均匀。细胞外液中谷氨酸浓度为10 mmol/L时，电压钳检测到稳定的细胞株，空载体组未检测到电信号，而GluA 1过表达组可检测到5-40 pA的电信号。结论成功建立了稳定表达GluA 1亚基的HEK 293 T单克隆细胞株。
Objective To build a cell line stably expressing alpha-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.