Effect of Antisense MBD1 Gene Eukaryotic Expression Plasmid on Expression of MBD1 Gene in Human Biliary Tract Carcinoma Cells / 华中科技大学学报（医学）（英德文版）

Hypermethylation of the promoter region is one of the major mechanism of tumor suppressor gene inactivation. In order to provide a research tool for the study on the function of MBD1gene in DNA methylation and tumorigenesis, antisense MBD1 gene eukaryotic expression plasmid was constructed and transfected into human biliary tract carcinoma cell line QBC-939 to observe its effect on the expression of MBD1 mRNA and protein by using RT-PCR and FCM respectively. Following the transfection, the mRNA level of MBD1 gene decreased from 0. 912±0. 022 to 0. 215±0.017, and the protein level of MBD1 gene also decreased from (80.19±5.05) % to (35.11±4.05)%. There were very significant differences in the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukary otic expression plasmid transfection group (P＜0.01). It was suggested that transfection with the antisense MBD1 gene eukaryotic expression plasmid can significantly reduce the expression level of MBD1 gene in QBC-939, and this study may provide a valid tool for the investigation of the function of MBD1 gene and its role in biliary tract carcinoma.

Construction and identification of eukaryotic expression vector of antisense MBD1 gene fragment / 中国普通外科杂志

Objective To construct eukaryotic expression vector of antisense MBD1 gene fragment and to provide a tool for studying MBD1 gene function. Methods PCR primers were designed according to the coding sequence of MBD1 gene. Xba I and Kpn I recognition sequences and cutting sites were added to the 5' end of the sense and antisense primer respectively. The 342 bp specific PCR fragment was obtained from the cDNA of biliary tract carcinoma cell line QBC-939 using RT-PCR, the purified PCR fragment was then inserted reversely into the multiple cloning site of eukaryotic expression vector pcDNA3. 1 ( + ). The constructed recombinant plasmid was identified by PCR confirmation, Xba I and Kpn I double enzyme digestion and DNA sequencing. Results The 322 bp specific DNA band was obtained by PCR, Xba I and Kpn I double digestion produced a 327 bp and a 5. 4 kb DNA band which represent the inserted target gene fragment and the vector respectively. The sequencing result confirmed that the sequence of inserted fragment was correct. Conclusion The eukaryotic expression vector of antisense MBD1 gene fragment was constructed successfully by using gene cloning technique. It will be a useful tool for studying MBD1 gene function in DNA methylation and tumorigenesis.