昆蟲細胞專一之四環黴素基因誘導系統
指導老師： 吳定峰
專題學生： 胡少卜
四技生物科技系四年甲班
1. Introduction
The most successful inducible system 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 while pTRE plasmid
contains a tetracyline-responsive element (TRE) composed of seven copies of tet operators
upstream of human cytomegalovirus immediate early (IE) mini-promoter (PminiCMV) which
lacks the enhancer region and is required to be activated by the AD domain of tTA. The gene
of interest will be cloned under the control of PminiCMV. In the absence of tetracycline,
multiple tTAs bind to TRE through the tetR domains and using the AD domains activate the
transcription of pTRE plasmid. In contrast, tetracycline will prevent the tTA from binding to
TRE in the presence of tetracycline because tTA has the higher affinity for tetracycline than
for TRE.
In this way the gene can be turned on/off by the tetracycline. However, Wu et. al. (2000)
reported that the CMV promoter of Tet-off system is not functional in the sf21 cells and the
induction rate of Tet-off system can be increased to 258-fold in TN368 cells by replacing the
PminiCMV with p10 promoter of Autographa californica multiple nuclear polyhedrosis virus
(AcMNPV). However, this induction rate is not compared to over 10,000-fold induction rate
when the Tet-off system is applied in the mammalian cells (Gossen et. al., 1992; Baron et. al.,
1997). In order to improve the induction rate of Tet-off system in the insect cells, it may be
feasible that the PminiCMV of pTet-off is replaced with the strong insect-specific promoters and
therefore more tTAs will be synthesized in the insect cells to stimulate the promoters.
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Lo et. al. (2001) reported that a novel enhancer present in the upstream region of
polyhedrin gene of AcMNPV or pAcUW21 baculovirus transfer vector (BD PharMingen),
which is used for the construction of recombinant AcMNPV can strongly activate the p10
promoter, PminiCMV and Drosaphila hsp70 promoter. This enhancer region encompasses the
ORF4, ORF5 and lef2 genes of AcMNPV and is called as the polyhedrin upstream (pu)
activator sequence. Deletion of each of ORF4, ORF5 and lef2 genes abolishes the function of
the pu sequence. Besides, the homologous region (hr) of AcMNPV and the pu sequence has
the synergistic activation effect on the PminiCMV. The hrs, which intersperse within the viral
genomes (Ayres et. al., 1994) have been proved to serve as the enhancers for early gene
trancription (Choi et. al., 1995).
2. Motivation
In this project, we replaced the CMV promoter of pTet-off with the PminiCMV hr or pu
sequence cloned downstream or upstream. Besides, the PminiCMV of pTRE was replaced by
the pag 1 gene promoter (-312/+29 region) of HZ-1 virus.
3. Results and discussion
Construction of modified pTet-off plasmids
The mini-CMV promoter, pu sequence and hr5 region were amplified with PCR from
pTRE2, pAcUW21 (Pharmingen) and hr5 respectively using the primers with appropriate
restriction enzyme sites. The sequences of the primers were listed in table 1. The sequences
of PCR products were confirmed by DNA autosequencing provided by the National
Cheng-Kung University DNA sequencing custom service. The enzyme-digested PCR
products were cloned into the appropriate sites of pTet-off as indicated in figure 1 to
construct the pTetCMVm, pTethrCMVm, pTetCMVmP and pTethrCMVmP.
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Evaluation of the induction rate of the modified Tet-off system in the sf9 cells
Each modified pTet-off was cotransfected with pTRE/-312/+29 into sf9 cells to evaluate
the induction rate, which contains the luciferase gene under the control of –312/+29 region
of HZ-1 virus pag 1 gene. As indicated in figure 2, the induction rates of pTethrCMVm,
pTetCMVmP and pTethrCMVmP all showed about 2-fold. That was compared to the
induction rate of transient transfection with only pTRE/-312/+29. This result suggested that
the pu and hr5 could not improve the 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. Western blot
hybridization may be performed to examine if tTA production was increased in sf9 cells by
the pu or hr5. In addition, the transient transfection may not be suitable for the examination
of the pu or hr5 effect on the production of tTA in sf9 cells.
4. Reference
Ayres, M. D., Howard, S. C., Kuzio, J., Lopez-Ferber, M., and Possee, R.D. (1994). The
complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 202,
586-605
Baron, U., Gossen, M., and Bujard, H. (1997). Tetracycline-controlled transcription in
eukaryotes: novel transactivators with graded transactivation potential. Nucleic acids Res. 25,
2723-2729.
Choi J., and Guarino L. A. (1995). The baculovirus transactivator IE1 binds to viral enhancer
elements in the absence of insect cell factors. J. Virol. 69, 4548-4551.
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. Biol.
Chem., 277:5256-64.
Gossen, M., and Bujard, H. (1992). Tight control of gene expression of in mammalian cells
by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. 89: 5547-5551.
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Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., and Bujard, H. (1995).
Transcriptional activation by tetracycline in mammalian cells. Science 268: 1766-1769.
Wu T.-Y., Liono, L., Chen, S.-L., Chen, C.-Y., and Chao, Y.-C. (2000). Expression of highly
controllable genes in insect cells using a modified tetracycline-regluated gene expression
stsyem. J. biotechnology 80, 75-83.
Table 1. The oligonucleotide sequences used in this study
Name
CMVmini
Sequence(5’-3’)
gggactagTTAGGCGTGTACGGTGGGAGG
ggggatccGCTGGATCGGTCCCGGTGTC
pu
gggaagcttCCTACAACTCCCCGCCCGCG
gggaagcttTCAATAATTACAAATAGGATT
GAGACC
hr5
gggactagtGCTTTACGAGTAGAATTCTAC
GCGgggactagtGTTTTACGCGTATTCTACCCG
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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 with pTRE2/-312/+29 plus pTethrCMVm;
lane 3, pTRE2/-312/+29 plus pTethrCMVm in presence of doxycycline; lane 4,
pTRE2/-312/+29 plus pTetCMVmP; lane 5, pTRE2/-312/+29 plus pTetCMVmP in presence
of doxycycline; lane 6, pTRE2/-312/+29 plus pTethrCMVmP; lane 7, pTRE2/-312/+29
plus pTethrCMVmP in presence of doxycycline; lane 8, pTRE2/-312/+29 plus pTetCMVm;
lane 9, pTRE2/-312/+29 plus pTetCMVm in presence of doxycycline; lane 10,
pTRE2/-312/+29; lane 11, pTRE2/-312/+29 in presence of doxycycline.
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