[A preliminary study on construction of targeting double suicide gene therapy vector pcDNA3.1 (-) Cp-CD-TK].
OBJECTIVE
To construct the targeting double suicide gene therapy vector pcDNA3.1 (-) Cp-CD-TK driven by carcino-embryonic antigen promoter (CEA promoter, Cp), and to investigate whether the vector could control the expression of CD-TK gene specificity and impact on proliferation of CEA positive colon cancer cells.


METHODS
Three kinds of target gene, Cp, CD, and TK were obtained by PCR, the products were double digested and inserted into pcDNA3.1 (-), then the targeting vector pcDNA3.1 (-) Cp-CD-TK was transfected into CEA positive human colon cancer SW480 cells and CEA negative Hela cells, the expression of CD-TK was examined by RT-PCR. The sensitivities of SW480 cells transfected with pcDNA3.1 (-) Cp-CD-TK to pro-drug 5-Fluorocytosine (5-Fc) and Ganciclovir (GCV) were detected by MTT analysis.


RESULTS
Recombinants with Cp, CD and TK insert were obtained. Constructed targeting gene therapy vector pcDNA3.1 (-) Cp-CD-TK was confirmed by gel electrophoresis and sequencing. RT-PCR analysis further confirmed CD-TK gene was expressed in SW480 cells and was not expressed in CEA negative Hela cells. MTT assay demonstrated that SW480 cells transfected with targeting vector pcDNA3.1 (-) Cp-CD-TK were sensitive to pro-drug 5-Fc and GCV.


CONCLUSIONS
Targeting double suicide gene therapy vector pcDNA3.1 (-) Cp-CD-TK was constructed correctly. The vector could make CD-TK gene express specifically in CEA positive cells for the purpose of targeting killing colon cancer.
Luo
Qi
Q
Department of General Surgery, Zhongshan Hospital, Xiamen University, Xiamen 361004, China.
Xiong
Yu
Y
Lu
Yi-Zhuo
YZ
D002272:Carcinoembryonic Antigen / Q000235:genetics*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000628:therapy; D043525:Cytosine Deaminase / Q000235:genetics / Q000502:physiology; D041722:Genes, Transgenic, Suicide / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics / Q000502:physiology
D002272:Carcinoembryonic Antigen; D013937:Thymidine Kinase; D043525:Cytosine Deaminase