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The novel insect cell-specific gene expression inducible system AcMNPV enhancer (hr or pu region). Our results showed that the induction rate could not be increased even in the presence of hr or pu region. The induction rate is about 2-fold. Key words : Tet-off system
Cause and goal
used in the mammalian cells is the Tet-off system. In this system, tTA is a fusion protein of VP16 activation domain (AD) of herpes simplex virus and wild type tetracycline repressor (tetR) which recognizes the tet operator (Gossen et. al., 1992; Gossen et. al., 1995). pTet-off is a tTA expression plasmid composed of seven copies of tet operators immediate early (IE) mini-promoter (PminiCMV) required to be activated by the AD domain of Abstract
tTA. The gene of interest will be cloned Tet-off system has been successfully applied under the control of PminiCMV. In the absence in mammalian cells with the induction rate of tetracycline, multiple tTAs bind to TRE over 10,000-fold. However, the induction through the tetR domains and using the AD rate is very low when Tet-off system is used domains activate the transcription of pTRE in the insect cells probably due to the low plasmid. In contrast, tetracycline will prevent transcriprional activity of CMV promoter the tTA from binding to TRE in the presence that drives the tTA gene. In this project, we of tetracycline because tTA has the higher replace the CMV promoter controlling the affinity for tetracycline than for TRE. In this tTA gene with the mini-CMV promoter way the gene can be turned on/off by the whose activity will be enhanced by the tetracycline. However, Wu et. al. (2000) reported that the CMV promoter of Tet-off hr or pu sequence cloned downstream or system is not functional in the sf21 cells and upstream. Besides, the PminiCMV of pTRE was the induction rate of Tet-off system can be replaced by the pag 1 gene promoter increased to 258-fold in TN368 cells by (-312/+29 region) of HZ-1 virus. replacing the PminiCMV with p10 promoter of Autographa californica multiple nuclear Results and Discussion
this induction rate is not compared to over Construction of modified pTet-off plasmids
10,000-fold induction rate when the Tet-off The mini-CMV promoter, pu sequence system is applied in the mammalian cells and hr5 region were amplified with PCR (Gossen et. al., 1992; Baron et. al., 1997). In from pTRE2, pAcUW21 (Pharmingen) and
order to improve the induction rate of Tet-off hr5 respectively using the primers with system in the insect cells, it may be feasible appropriate restriction enzyme sites. The that the PminiCMV of pTet-off is replaced with sequences of the primers were listed in table the strong insect-specific promoters and 1. The sequences of PCR products were therefore more tTAs will be synthesized in confirmed by DNA autosequencing provided the insect cells to stimulate the promoters. Lo et. al. (2001) reported that a novel DNA sequencing custom service. The enhancer present in the upstream region of enzyme-digested PCR products were cloned polyhedrin gene of AcMNPV or pAcUW21 into the appropriate sites of pTet-off as baculovirus transfer vector (BD PharMingen), indicated in figure 1 to construct the which is used for the construction of pTetCMVm, pTethrCMVm, pTetCMVmP recombinant AcMNPV can strongly activate and pTethrCMVmP. the p10 promoter, PminiCMV and Drosaphila hsp70 promoter. This ehnacer region Evaluation of the induction rate of the
encompasses the ORF4, ORF5 and lef2 genes modified Tet-off system in the sf9 cells
of AcMNPV and is called as the polyhedrin Each modified pTet-off was upstream (pu) activator sequence. Deletion of cotransfected with pTRE/-312/+29 into sf9 each of ORF4, ORF5 and lef2 genes cells to evaluate the induction rate, which abolishes the function of the pu sequence. contains the luciferase gene under the control Besides, the homologous region (hr) of of –312/+29 region of HZ-1 virus pag 1 gene. AcMNPV and the pu sequence has the As indicated in figure 2, the induction rates synergistic activation effect on the PminiCMV. of pTethrCMVm, pTetCMVmP and The hrs, which intersperse within the viral pTethrCMVmP all showed about 2-fold. genomes (Ayres et. al., 1994) have been That was compared to the induction rate of proved to serve as the enhancers for early transient transfection with only gene trancription (Choi et. al., 1995). pTRE/-312/+29. This result suggested that In this project, we replaced the CMV the pu and hr5 could not improve the promoter of pTet-off with the PminiCMV with induction rate of Tet-off system in sf9 cells. It was possible that hr5 and pu did not increase the tTA production in sf9 cells. Gossen, M., and Bujard, H. (1992). Tight Western blot hybridization may be performed control of gene expression of in mammalian to examine if tTA production was increased cells by tetracycline-responsive promoters. in sf9 cells by the pu or hr5. In addition, the Proc. Natl. Acad. Sci. 89: 5547-5551. transient transfection may not be suitable for the examination of the pu or hr5 effect on the Muller, G., Hillen, W., and Bujard, H. (1995). Transcriptional activation by tetracycline in Self evaluation
mammalian cells. Science 268: 1766-1769. pu and hr5 seemed not to increase the tTA production in the sf9 cells. We may Wu T.-Y., Liono, L., Chen, S.-L., Chen, C.-Y., try the western blot hybridization to evaluate and Chao, Y.-C. (2000). Expression of highly the amount of tTA in sf9 cells. The stable controllable genes in insect cells using a Reference
Lopez-Ferber, M., and Possee, R.D. (1994). Table 1. The oligonucleotide sequences used The complete DNA sequence of Autographa in this study californica nuclear polyhedrosis virus. Name Sequence(5’-3’) Baron, U., Gossen, M., and Bujard, H. (1997). Tetracycline-controlled transcription in eukaryotes: novel transactivators with graded transactivation potential. Nucleic acids Res. baculovirus transactivator IE1 binds to viral enhancer elements in the absence of insect Lo H. R,. Chou C. C., Wu T. Y., Yuen J. P., and Chao Y. C. (2001). Novel baculovirus DNA elements strongly stimulate activities of exogenous and endogenous promoters. J. Figure 1. The modified pTet-off.
Figure 2. The results of the transient
transfection for the modified Tet-off system.
Lane 1, sf9; lane 2, sf9 transiently transfected pTRE2/-312/+29 plus pTetCMVmP; lane 5, pTRE2/-312/+29 plus pTethrCMVmP; lane 7, pTRE2/-312/+29 plus pTethrCMVmP in The novel insect cell-specific gene expression inducible system

Source: http://eshare.stust.edu.tw/ir/2009_1/2009_1_20e73d26.pdf

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