Construction and identification of recombinant vectors carrying herpes simplex virus thymidine kinase and cytokine genes expressed in gastric carcinoma cell line SGC7901.
OBJECTIVE
To construct and identify the recombinant vectors carrying herpes simplex virus thymidine kinase (HSV-TK) and tumor necrosis factor alpha (TNF-alpha) or interleukin-2 (IL-2) genes expressed in gastric carcinoma cell line SGC7901.


METHODS
The fragments of HSV-TK, internal ribosome entry sites (IRES) and TNF-alpha or IL-2 genes were inserted in a TK-IRES-TNF-alpha or TK-IRES-IL-2 order into pEGFP-N(3) and pLXSN to generate the therapeutic vectors pEGFP-TT, pEGFP-TI, pL(TT)SN and pL(TI)SN respectively, which were structurally confirmed by the digestion analysis of restriction endonuclease. The former two plasmids were used for the transient expression of recombinant proteins in the target cells while pL(TT)SN and pL(TI)SN were transfected into SGC7901 cells by lipofectamine for the stable expression of objective genes through G418 selection. The protein products expressed transiently and stably in SGC7901 cells by the constructed vectors were confirmed by fluorescent microscopy and Western blot respectively.


RESULTS
The inserted fragments in all constructed plasmids were structurally confirmed to be consistent with that of the published data. In the transient expression, both pEGFP-TT and pEGFP-TI were shown expressed in nearly 50% of the transfected SGC7901 cells. Similarly, the G418 selected vectors PL(TT)SN and PL(TI)SN were confirmed to be successful in the stable expression of the objective proteins in the target cells.


CONCLUSIONS
The constructed recombinant vectors in the present study that can express the suicide gene TK in combination with cytokines genes may serve as the potential tools to perform more effective investigations in future for the gene therapy of gastric carcinoma.
Zhang
Jian-Hua
JH
Department of Biomedical Engineering, School of Life Science and Technology, Xi'an Jiaotong University, 28 West Xianning Road, Xi'an 710049, Shaanxi Province, China.
Wan
Ming-Xi
MX
Yuan
Jia-Ying
JY
Pan
Bo-Rong
BR
D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods; D004274:DNA, Recombinant; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D007376:Interleukin-2 / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D018139:Simplexvirus / Q000235:genetics*; D013274:Stomach Neoplasms; D013937:Thymidine Kinase / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D004274:DNA, Recombinant; D007202:Indicators and Reagents; D007376:Interleukin-2; D008164:Luminescent Proteins; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase