| In recent years the specific promoters are often used to control the therapeutic gene expression by regulation of transcription in order to achieve tumor specific target in cancer gene therapy. A kind of tissue specific promoters, osteocalcin promoter, has been used in osteosarcoma gene therapy. However, it maybe results in severe side effects in clinical therapy because the osteocalcin promoter's activity in nomal osteocytes is the same as in osteosarcoma cells. It has been already confirmed that the activity of human telomerase reverse transcriptase (hTERT) rise in approximately 90% human tumors whereas most nomal cells do not express the activity and it becomes a hotspot in transcriptional target of cancer gene therapy. But only one natural promoter can hardly reach the requests of cancer gene therapy, which demand high activity and selectivity, so experts try to customize the synthetic promoter with positive or negative elements from a variety of different promoter/enhancers. Based on the moleculebiological characteristic of osteosarcoma that the level of c-Myc expression is high and hTERT promoter activity is identified, we modify the hTERT core promoter with MYC-responsive elements to improve its activityand specificity in osteosarcoma.Virus-directed enzyme prodrug therapy (VDERT) has been popular in cancer gene therapy due to its potential foe tumor-specific cytotoxicity. To gain the breakthrough in osteosarcoma gene therapy, we construct replicate -deficiency adenoviruses with Fiyl or luciferase driven by hTERT core promoter modified with MYC-responsive elements utilizing AdMax Kit D containing Cre /loxP system and assess the activity of synthetic promoters by semi-quantitive analysis. It has been not found the similarity reports in the world.1. Shuttle plasmids constructedConstruct the hTERT core promoter modified with MYC-responsive elements DNA sequence according to the design, then clone them with therapentic gene Fcyl or reporter gene luciferase to the shuttle plasmids provided by AdMax Kit D respectively.2. Recombine and replicate replicate-deficiency adenovirusCo-transfect the HEK 293 cells with rescue plasmid and shuttle plasmid containing Fcyl or luciferase driven by hTERT core promoter modified with MYC-responsive elements. 7-10 days after co-transfection the cytopathic effect (CPE) appear, that is, infected cells typically remain intact but round up and may detach from the plate. Collect virus supernatant.and then infect HEK 293 cells with virus supernatant. After 3-4 days when CPE appear, collect virus supernatant.3. Recombinant adenovirus identifiedThe DNA were extracted from the virus lysate and detected by PCR using Fcyl or luciferase specific primers. The results show that all the 4 viruses of the Fcyl group can amplify the 122bp targeted fragment and all the 4 viruses of the luciferase group can amplify the 312bp targeted fragment. The result suggests9we get the right adenoviruses as our design.4. Determine recombinant adenovirus titerPlaque assay is a biological assay to determine adenovirus titer. We get the quantity of recombinant adenovirus by the formula:Titer (pfu/ml) = # Plaques / d X v d = dilution factor v = volume of diluted virus added to the well5. Assess the activity of synthetic promoters by semi-quantitive analysis (Evaluate Fcyl mRNA in MG63 cells infected with recombinantadenoviruses by RT-PCR)Adjust the adenoviral titers of Fcyl group to one standard and then infect MG63 cells (human osteosarcoma cell) respectively. After 48 hours isolate the total RNA of cells as samples to RT-PCR using primers specific to Fcyl and ?-actin regarded as internal control. The difference of promoters activities can be assessed through the light density ratio of the RT-PCR products of Fcyl to & -actin mRNA by gel analysis system. The results indicates that the activity of synthetic promoter is significant higher than the natural one, however, the synthetic promoter activity does not al...
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