pRetroX-Tet3G plasmid vector多西环素诱导调控逆病毒表达质粒载体 BioVector NTCC质粒载体菌种细胞基因保藏中心

pRetroX-Tet3G plasmid vector多西环素诱导调控逆病毒表达质粒载体

BioVector® Retro-X™ Tet-On®3G Inducible Expression System
Summary
The Tet-On 3G Systems are inducible gene expression systems for mammalian cells. Target cells that
express the Tet-On 3G transactivator protein and contain a gene of interest (GOI) under the control of a
TRE3G promoter (PTRE3G or PTRE3GV) will express high levels of your GOI, but only when cultured in the
presence of doxycycline (Dox) (Figure 1).
Figure 1. The Tet-On 3G Systems allow inducible gene expression in the presence of Dox.
B. Elements of Retro-X Tet-On 3G
Tet-On 3G Transactivator Protein
Based on the transcriptional regulators described by Gossen & Bujard (1992), Gossen et al. (1995), and
Urlinger et al. (2000), Tet-On 3G is a modified form of the Tet-On Advanced transactivator protein which
has been evolved to display far higher sensitivity to doxycycline (Zhou et al. 2006).
PTRE3GV Inducible Promoter
The inducible promoter PTRE3G provides for very low basal expression and high maximal expression after
induction (Loew et. al., 2010). It consists of 7 repeats of a 19 bp tet operator sequence located upstream
of a minimal CMV promoter. PTRE3GV is a version of PTRE3G that was modified at Takara Bio for higher
performance in retroviruses and lentiviruses. In the presence of Dox, Tet-On 3G binds specifically to
PTRE3GV and activates transcription of the downstream GOI. PTRE3GV lacks binding sites for endogenous
mammalian transcription factors, so it is virtually silent in the absence of induction.
Retro-X Universal Packaging System
The Retro-X Universal Packaging System is our premium retroviral packaging system, featuring the high
titer GP2-293 packaging cell line. All four commonly used envelopes are supplied on separate vectors,
including VSV-G, eco, ampho and 10A1, to allow you to choose the tropism that is most appropriate for
your target cells. The envelope vector is cotransfected with your retroviral expression vector, and high
titers of pantropic, ecotropic, amphotropic, or dualtropic virions can be obtained in less than 48 hr.
C. Doxycycline
Doxycycline is a synthetic tetracycline derivative that is the effector molecule for the Tet-On and TetOff® Systems. When bound by Dox, the Tet-On 3G protein undergoes a conformational change that
allows it to bind to tet operator sequences located in the PTRE3G promoter (Figure 1). The Dox
concentrations required for induction of Tet-On Systems are far below cytotoxic levels for either cell
culture or transgenic studies, and Tet-On 3G responds to even lower concentrations than its predecessors
(Zhou et al., 2006). Note that Tet-On Systems respond well only to doxycycline, and not to tetracycline
(Gossen & Bujard, 1995). The half-life of Dox in cell culture medium is 24 hr. To maintain continuous
inducible GOI expression in cell culture, the medium should be replenished with Dox every 48 hr.
List of Components
Store the GP2-293 Packaging Cell Line at –196°C and all other components at –20°C.
Retro-X Tet-On 3G Inducible Expression System (Cat. No. 631188).
• Retro-X Tet-On 3G Vector Set
o pRetroX-Tet3G Vector (10 µg)
o pRetroX-TRE3G Vector (10 µg)
o pRetroX-TRE3G-Luc Control Vector (10 µg)
• Retro-X Universal Packaging Vector Set
o p10A1 Vector (20 μg)
o pAmpho Vector (20 μg)
o pEco Vector (20 μg)
o pVSV-G Vector (20 μg)
o pQCLIN Retroviral Vector (20 μg)
• GP2-293 Packaging Cell Line (1 ml)
• Xfect™ Transfection Reagent (100 rxns)
• Tet System Approved FBS, US-Sourced (50 ml)



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