892626b04100

Utilization of the insect-specific tetracycline-inducible expression system for the development Cause and goal
developed for biopesticide to shorten the long killing time of wild type baculovirus. constitutively expressed toxin genes are not suitable to be used as the biopesticides due to the difficulty of mass production. Therefore, allowing tight and specific regulation of toxin gene expression are important for the Abstr act
development of engineered biopesticides. In previous studies, 30 fold of induction Tetracycline inducible expression system rate could be observed when tet-off system was applied in the sf9 cells. However, over 1000 fold of induction rate can be measured mammalian cells. Therefore, in this project tet-off system was modified to be insect mammalian cells. However, in our previous studies we detected only 30 fold of induction pTRE of tet-off system was replaced by the when the tet-off system was employed in the promoter of pag 1 gene of HZ-1 virus. Over sf21 cells (1). In order to increase the 1000 fold of induction rate could be observed induction fold of tet-off system in the insect when the insect-specific tet-off system was cell, the residual basal activity has to be used in sf9 cells. The result of this project reduced and a strong insect-specific promoter may be used in the tet-off system. Lee et. al. (1998) (2) and Chen et. al. (2000) (3) Key wor ds : Tet-off system
region) of pag1 gene of insect-specific HZ-1 virus shows the strong activity in the insect KpnI/BamHI-linearized pTRELuc to replace cells and can be significantly repressed by the –312/ –90 region of the pag 1 promoter. pTRE/pag –90/+29Luc (Fig. 1). To construct In this project, two insect-specific TRE pag –312/-90-TRE-pag–90/+29 promoter, the XhoI-digested pag –312/-90 PCR fragment miniCMV promoter and the pag-312/-90 was upstream of pag promoter. In the presence of p(pag –312/-90)/TRE/pag –90/+29Luc (Fig. tetracycline, tetracycline will prevent the tTA 1). To construct TRE-pag –312/+29 promoter, the pag –312/+29 PCR fragment was ligated with the BamHI/KpnI-restricted pTRELuc inhibited by pag-312/-90. In the absence of through the tetR domains and may abolish the repression of pag-312/-90 to activate the –90/+29 region of pag1 gene. In this way, The regulation of pag –312/-90-TRE-pag–
90/+29 promoter by tetr acycline
significantly and high expression of the gene of interest is produced by the pag 1 promoter. pag –312/-90-TRE-pag –90/+29 promoter by the tetracycline (Tc), the p(pag –312/-90)/ Results and Discussion
TRE/pag –90/+29Luc was cotransfected into The constr uction of insect-specific TRE
absence of Tc. As indicated in Fig. 3, the promoter s
luciferase activity was 183-fold higher in the absence of Tc than in the presence of 1 µg Tc were constructed. The pag –312/-90-TRE- pag –312/-90-TRE-pag–90/+29 promoter pag –312/-90 fragment upstream of TRE and could be regulated by Tc. However, in the the pag –90/+29 fragment replacing the absence of ptTA and Tc the pag –312/-90-TR E-pag –90/+29 promoter alone could express The TRE-pag –312/+29 promoter contains the comparable luciferase activity (Fig. 3, the pag –312/+29 fragment replacing the column C). This result was required to be elucidated. Consistent with the previous contains a luciferase gene under the control results, the luciferase activity of pTRELuc the sf9 cells (Fig. 3, columns F, G and H). promoter of pTRELuc with pag –90/+29, the KpnI/BamHI-digested pag –90/+29 PCR performed three times. The activation folds for three experiments were 183, 276 and 25 fold respectively. It was likely that the sf9 function like tTA protein to activate the cells were not healthy when the experiment TRE/-90/+29 promoter and can be regulated by Tc. In order to test our hypothesis, the pag –312/-90-TRE-pag –90/+29 promoter The regulation of TRE-pag –312/+29
was measured in the presence of Tc without promoter by tetr acycline
ptTA present. Results demonstrated that the pag –312/-90-TRE-pag –90/+29 promoter pTRE/pag –312/+29Luc was cotransfected alone was reduced 160 fold by Tc (Fig. 5, into sf9 cells with ptTA in the presence or column A and B), suggesting some protein absence of Tc. As demonstrated in Fig. 4, the present in the sf9 cells may act like tTA luciferase activity was 2276-fold higher in protein. This phenomenon was required to be the absence of Tc than in the presence of 1 µg Tc /ml medium, suggesting that the TRE-pag–312/+29 Self evaluation
regulated by Tc. The activity and activation be observed when the insect-specific tet-off much higher than those of pag –312/-90 / system was used in sf9 cells. This system is TRE / pag –90/+29 promoter. Like pag –312 worthwhile to be studied for the application absence of ptTA and Tc the TRE-pag –312/+ Reference
1. Chen, H. H., Lee, S. T., Chen, C. T., and activation folds for two experiments were 2276 and 867 fold respectively. It was likely that the sf9 cells were not healthy enough Agricultural Chemistry, 2, 220-225.
2. Chao, Y. C., Lee, S. T., Chang, M. C., Chen, H. H., Chen, S. S., Wu, T. Y., Liu, F. H., Hsu, E. L., and Hou, R.F. 1998. A Effect of Tc on pag –312/-90-TRE-pag –90
/+29 promoter in the absence of ptTA
persistent Hz-1 viral infection. Journal of In the previous result, the pag –312/-90- 3. Chen, H. H., Tsai, F. Y., and Chen, S. T. factors for its regulatory effect. Journal of General Virology. 82, 313-320.
Figure 4. The regulation of TRE-pag-312/
+29 promoter by Tc
Xho I Xho I Kpn IBamH I Figur e 1. The pag-312/-90-TRE-pag-90/+29
Figur e 2. The TRE-pag-312/+29 pr omoter
Figure 3. The regulation of pag-312/-90
-TRE pag-90/+29 promoter by Tc

Source: http://ir.chna.edu.tw/bitstream/310902800/2011/1/NSC89-2626-B-041-001.pdf