title: [Establishment of esophageal squamous carcinoma cell lines stably over-expressing LLGL2].
abstract: Objective To prepare a lentiviral vector expressing LLGL2 and establish KYSE450 and TE-1 cell lines for the stable expression of LLGL2. Methods The full-length LLGL2 sequence was amplified by high-fidelity PCR, and then it was inserted into pCDH-CMV-IRES-GFP-EF1-Puro vectors. The recombinant plasmid was confirmed by double enzyme digestion and sequencing. After co-infection of pCDH-CMV-LLGL2-IRES- GFP-EF1-Puro with vesicular stomatitis virus glycoprotein (VSVG) and PHR into HEK293T cells, the lentivirus was harvested and used for infecting esophageal squamous cell carcinoma cell lines including KYSE450 and TE-1 cells. These two cell lines infected with the lentivirus were screened with puromycin, and the stable cell lines were further confirmed with green fluoresence and Western blotting. Results Dual-enzyme digestion and sequencing confirmed that the pCDH-CMV-LLGL2-IRES-GFP-EF1-Puro vector, a lentiviral expression vector for the overexpression of LLGL2, was successfully constructed through high-fidelity PCR and ligation. Western blotting showed the increased expression level of LLGL2 protein in KYSE450 and TE-1 stable cell lines compared with the controls. Conclusion The experiment successfully established KYSE450 and TE-1 stable cell lines for the overexpression of LLGL2.
authors.affiliation: Dongfang Hospital, Xiamen University, Fuzhou 350025, China.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Hu
authors.affiliation: PLA Institute for Laboratory Medicine, Fuzhou General Hospital of Nanjin Military Area Command, Fuzhou 350025, China.
authors.forename: Fu'an
authors.initials: F
authors.lastname: Xie
authors.affiliation: Dongfang Hospital, Xiamen University, Fuzhou 350025, China.
authors.forename: Xiaoqian
authors.initials: X
authors.lastname: Hong
authors.affiliation: Dongfang Hospital, Xiamen University, Fuzhou 350025; PLA Institute for Laboratory Medicine, Fuzhou General Hospital of Nanjin Military Area Command, Fuzhou 350025, China. *Corresponding author, E-mail: liukuancan@163.com.
authors.forename: Kuancan
authors.initials: K
authors.lastname: Liu
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000378:metabolism; D004938:Esophageal Neoplasms / Q000235:genetics* / Q000378:metabolism; D000077277:Esophageal Squamous Cell Carcinoma; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D003598:Cytoskeletal Proteins; C526122:llgl2 protein, human