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Objective To clone human Thocl gene and construct the eukaryotic expression vector pThocl-DsRed2-N1 , to observe the co-localization of Thocl with voltage-gated chloride channel 3 (C1C-3) in human cervical cancer HeLa cells, and to analyze whether C1C-3 is involved in the cell transcription. Methods Thocl coding sequence was amplified by RT-PCR and cloned into the pDsRed2-Nl vector to construct the eukaryotic expression vector pThocl-DsRed2-Nl , which was then identified by PCR, enzyme digestion (Sac 1 and RamH 1 ) , and gene sequencing. HeLa cells in the logarithmic phase were divided into 3 groups: the transfection group, empty transfection group, and control group, which were transfected with pThocl -DsRed2-Nl plasmid, pDsRed2-Nl plasmid, and lipidosome by Lipofectamine 2000, respectively. At 48 h, the gene expression was observed under inverted fluorescence microscope. The co-localization of Thocl with C1C-3 was detected by immunofluorescence in the the transfection group. Results PCR results showed that there was a 2. 1 kb target band, and meanwhile, 4. 7 kb and 2. 1 kb bands were obtained by enzyme digestion. Sequencing proved that the sequence and splicing order of Thocl gene was matched with Genebank, suggesting that the fluorescent eukaryotic expression vector pThocl-DsRed2-Nl was constructed successfully. Overt red fluorescence was observed in the empty transfection group. The red fluorescence intensity of the transfection group was between those of the empty transfection group and the blank control group, suggesting that Thocl was expressed in HeLa cells. C1C-3 was localized both in the nucleus and cytoplasm, mainly in nucleus. At the same time, Thocl was localized in the nucleus only, and yellow fluorescence was observed after we merged C1C-3 with Thocl , indicating that C1C-3 and Thocl were co-located in the nucleus. Conclusion The eukaryotic expression vector pThocl-DsRed2-Nl is successfully constructed, Thocl co-localizes with C1C-3 in the nucleus, suggesting that C1C-3 may be involved in the cell transcription process. [ABSTRACT FROM AUTHOR]

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