| Cancer is one of the major threats to public health in modern society. Gene therapy has been developing so rapidly and will bring about promise in conquering this severely life-threatening disease. The gene delivery system is the most important part of the overall gene therapy system.By now,adenovirus-based vectors have become widely used vehicles for cancer gene therapy.It has recently been shown that high levels of transgene expression in certain antigen-presenting cells significantly increase the response of the host immune system to that transgene, thereby limiting vector efficacy.For cancer therapy strategies that utilize adenoviral vectors to deliver cytotoxic genes to tumor cells, selectively restricting the expression of these transgenes to their target cells is clearly desirable. A strategy known as transcriptional targeting has been developed to overcome these problems. It utilizes tissue-specific promoters to express selectively the transgenesin target cells. However, selective activity of some promoters-that have been originally characterized as highly specific-was altered orlost when they were placed in the context of adenoviral genome. A hypothesis advanced to explain this phenomenon postulated that enhancer elements and/or cryptic transcription start sites within the adenoviral sequences surrounding the transgene expression cassette were interacting with the promoter elements to activate'illicit'transcription in nontarget cells. Enhancer activity has been reported for such Ad5 sequences as the inverted terminal repeats (ITR), E2 and E4 promoters, the pIX promoter and the E1A enhancer. The adenovirus type 5 genome contains two distinct enhancer elements located at the left end of the viral chromosome.The first element is repeated and specifically regulates region E1A transcription within infected cells. The second element is located between these repeated sequences and regulates transcription in cis of all early regions on the chromosome.Objective:Construct a series of recombinant adenovirus vectors that contains green fluoresent protein (GFP) To study the expression of specific gene downstream of the left inverted terminal repeat (ITR) in adenovectors by fluorometric assay.Methods :Construct a series of recombinant adenovirus vectors that contains green fluoresent protein (GFP) and hTERT promoter and CMV promoter and recombinant adenovirus vectors containing the same transgene but in different orientations. The recombinant adenovirus plasmids were digested with Pac I and transferred into 293A cells mediated by LiopFectamine 2000 to package recombinant adenovirus particles and then titrated using 50% tissue culture infective dose (TCID50) assay. All of the virus infected four tumor cell whith the same MOI and the green fluorescence was observed by fluorescent microscopy.Results:1 The recombinant adenovirus hTERT, rhTERT, CMV, rCMV and rGFP was successfully constructed and transferred into 293A cells with high titer.2 After All of the virus infected 293A cells,CNE cells.U-2OS cells,435 cells and Panc1 cells,the strong fluorescent can be observed in 293A cells and Panc1 cells.3 By statistical analysis, the fluorescent in each group does not exist significant difference.4 The faint fluorescent can be observed in the cell in which adenovirus absence of promoter.Conclusion:1 The enhancer elements of the left ITR in recombinant adenovirus significant effect the expression of the upstream gene and downstream gene but the influence does not exist significant difference.2 Studies by this experiment have also demonstrated that the left ITR contains cryptic transcription start sites. |
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