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제안 방법
- In order to determine whether tetO sequences mediate the stimulation of the gene expression by HCMV infection in the absence of tTA, we analyzed the e仟ect of the gene expression from the minimal promoter by removing the tetO sequences. A construct (pminBg) was made by deleting the seven direct repeats of the 42 bp tetO sequences from p7tetminBg.
- In order to determine whether the tTA-mediated activation system could be established in HCMV we evaluated the effect of tTA on the gene expression from the HCMV minimal promoter containing the tetO sequences. We constructed an indicator plasmid (p7tetminBg) which contained seven direct repeats of the 42 bp tetO sequences (Gossen and Bujard, 1992), the HCMV minimal promoter (Gossen and Bujardz 1992), 林밍ucuronidase, and the HSV-1 thymidine kinase poly A signal (Jones and Muzithras, 1991).
- In this study, we examined whether the tTA-mediated gene activation system could be applied to HCMV and HSV-1 replication cycles in order to control viral gene expression. The gene expression of the reporter gene, under the control of the minimal promoter containing the tetO sequences, was controlled by the presence of tTA.
이론/모형
- The resulting construct (pminBg) contained the HCMV minimal promoter, the P-glucuronidase gene, and the poly A signal. Either p7tetminBg or pminBg was transfected into HFF cells using the DEAE-dextran method. On HCMV infection, the gene activation of p7tetminBg was approximately 18 times higher than the gene activation of pminBg ("Table II).
- Cell extraction was performed 48 h post-transfection, and the assay was done, as described above. The total protein in the cell extracts was determined by the Bradford method as described in the kit (Bio-Rad).
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