四環(huán)素調控系統(tǒng) pRevTet-On

pRevTet-On

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.