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
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
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
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
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