pRevTet-On四環素誘導表達逆轉錄病毒系統質粒載體表達rtTA BioVector NTCC質粒載體菌種細胞基因保藏中心
Description:
pRevTet-On is a retroviral vector expressing the reverse tetracycline-controlled transactivator
(rtTA) from the CMV promoter. This vector is derived from pLNCX, a retroviral vector created
using elements of Moloney murine leukemia virus (MoMuLV) and Moloney murine sarcoma
virus (MoMuSV) as described (1). rtTA is a fusion of amino acids 1–207 of the reverse tet
repressor (rTetR) and the negatively charged C-terminal activation domain (130 amino acids)
of the VP16 protein of Herpes Simplex Virus. rTetR was derived from TetR and differs by four
point mutations, which are responsible for its opposite response to doxycycline. The 5' viral
LTR controls expression of the transcript that contains Ψ+ (the extended viral packaging signal)
and the neomycin resistance gene (Neor) for antibiotic selection in mammalian cells. rtTA is
derived from vectors described previously (2–4). pRevTet-On also includes the E. coli Ampr
gene for antibiotic selection in bacteria.
Use:
pRevTet-On can be used to establish stable Tet-On cell lines via retrovirus-mediated gene
transfer (5). Retroviral gene transfer allows the highly efficient transduction of virtually all
dividing cell types. The RevTet Systems are also suitable for establishing transgenic animals. In
combination with the pRev-TRE retroviral expression vector, a gene of interest can be inducibly
expressed at high levels in response to varying concentrations of the tetracycline derivative
doxycycline (Dox). rtTA binds to the Tet-response element (TRE), thus activating transcription in the presence of Dox. The response of rtTA to Dox is thus opposite to the response of
tTA. As Dox is removed from the culture medium, transcription from the inducible promoter
is turned off in a highly dose-dependent manner. pRevTet-On lacks the viral genes gag, pol,
and env, which are supplied by the packaging cell line. It can be transfected into a high titer
packaging cell line and thereby mediate production of infectious, replication-incompetent
retroviral particles (1, 6–7).The transcript produced by the pRevTet-On construct is recognized
by the viral structural proteins expressed in a packaging cell line and packaged into infectious
retroviral particles. Because the RNA transcript packaged in these particles does not contain
the viral genes, it cannot replicate in the target cells that it infects.
The level of induction in cell populations infected with this vector depends on the efficiency
of infection, the site of integration, and the titer of the virus. Viral supernatants with titers >105
cfu/ml should be produced to achieve high-level induction.
Location of features:
?  5' MoMuSV LTR: 1–589
?  Ψ+ (extended packaging signal): 659–1468
?  Neomycin resistance gene: 1512–2306
   Start codon: 1512–1514; stop codon: 2304–2306
?  CMV promoter (PCMV): 2654–3464
?  rtTA coding sequence: 3481–4488
?  3' MoMuLV LTR: 4551–5144
?  Ampicillin resistance (β-lactamase) gene: 7298–6438
   Start codon: 7298–7296; stop codon: 6440–6438
Propagation in E. coli:
?  Suitable host strains: DH5α, HB101, and other general purpose strains.
?  Selectable marker: plasmid confers resistance to ampicillin (50 μg/ml) to E. coli hosts.
?  E. coli replication origin: Col E1
?  Copy number: low copy
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