pRevTet-Off? is a retroviral vector expressing the tetracycline-controlled transactivator (tTA) 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). tTA is a fusion of amino acids 1–207 of the tet repressor (TetR) and the negatively charged C-terminal activation domain (130 amino acids) of the VP16 protein of Herpes Simplex Virus. The 5' viral LTR controls expression of the transcript that contains Y+ (the extended viral packaging signal), and the neomycin resistance gene (Neor) for antibiotic selection in mammalian cells. tTA is derived from vectors described previously (2, 3). pRevTet-Off also includes the E. coli Ampr gene for antibiotic selection in bacteria.

載體應用

pRevTet-Off can be used to establish stable Tet-Off cell lines via retrovirus-mediated gene transfer (4). 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 tetracycline (Tc) or Tc derivatives such as doxycycline (Dox). tTA binds to the Tet-response element (TRE), thus activating transcription in the absence of Tc or Dox. As Tc or Dox is added to the culture medium, transcription from the inducible promoter is turned off in a highly dose-dependent manner. pRevTet-Off 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, 5–6).The transcript produced by the pRevTet-Off 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.

0001 TTTGAAAGAC CCCACCCGTA GGTGGCAAGC TAGCTTAAGT AACGCCACTT TGCAAGGCAT 
0061 GGAAAAATAC ATAACTGAGA ATAGAAAAGT TCAGATCAAG GTCAGGAACA AAGAAACAGC 
0121 TGAATACCAA ACAGGATATC TGTGGTAAGC GGTTCCTGCC CCGGCTCAGG GCCAAGAACA 
0181 GATGAGACAG CTGAGTGATG GGCCAAACAG GATATCTGTG GTAAGCAGTT CCTGCCCCGG 
0241 CTCGGGGCCA AGAACAGATG GTCCCCAGAT GCGGTCCAGC CCTCAGCAGT TTCTAGTGAA 
0301 TCATCAGATG TTTCCAGGGT GCCCCAAGGA CCTGAAAATG ACCCTGTACC TTATTTGAAC 
0361 TAACCAATCA GTTCGCTTCT CGCTTCTGTT CGCGCGCTTC CGCTCTCCGA GCTCAATAAA 
0421 AGAGCCCACA ACCCCTCACT CGGCGCGCCA GTCTTCCGAT AGACTGCGTC GCCCGGGTAC 
0481 CCGTATTCCC AATAAAGCCT CTTGCTGTTT GCATCCGAAT CGTGGTCTCG CTGTTCCTTG 
0541 GGAGGGTCTC CTCTGAGTGA TTGACTACCC ACGACGGGGG TCTTTCATTT GGGGGCTCGT 
0601 CCGGGATTTG GAGACCCCTG CCCAGGGACC ACCGACCCAC CACCGGGAGG TAAGCTGGCC 
0661 AGCAACTTAT CTGTGTCTGT CCGATTGTCT AGTGTCTATG TTTGATGTTA TGCGCCTGCG 
0721 TCTGTACTAG TTAGCTAACT AGCTCTGTAT CTGGCGGACC CGTGGTGGAA CTGACGAGTT 
0781 CTGAACACCC GGCCGCAACC CTGGGAGACG TCCCAGGGAC TTTGGGGGCC GTTTTTGTGG 
0841 CCCGACCTGA GGAAGGGAGT CGATGTGGAA TCCGACCCCG TCAGGATATG TGGTTCTGGT 
0901 AGGAGACGAG AACCTAAAAC AGTTCCCGCC TCCGTCTGAA TTTTTGCTTT CGGTTTGGAA 
0961 CCGAAGCCGC GCGTCTTGTC TGCTGCAGCG CTGCAGCATC GTTCTGTGTT GTCTCTGTCT 
1021 GACTGTGTTT CTGTATTTGT CTGAAAATTA GGGCCAGACT GTTACCACTC CCTTAAGTTT 
1081 GACCTTAGGT CACTGGAAAG ATGTCGAGCG GATCGCTCAC AACCAGTCGG TAGATGTCAA 
1141 GAAGAGACGT TGGGTTACCT TCTGCTCTGC AGAATGGCCA ACCTTTAACG TCGGATGGCC 
1201 GCGAGACGGC ACCTTTAACC GAGACCTCAT CACCCAGGTT AAGATCAAGG TCTTTTCACC 
1261 TGGCCCGCAT GGACACCCAG ACCAGGTCCC CTACATCGTG ACCTGGGAAG CCTTGGCTTT 
1321 TGACCCCCCT CCCTGGGTCA AGCCCTTTGT ACACCCTAAG CCTCCGCCTC CTCTTCCTCC 
1381 ATCCGCCCCG TCTCTCCCCC TTGAACCTCC TCGTTCGACC CCGCCTCGAT CCTCCCTTTA 
1441 TCCAGCCCTC ACTCCTTCTC TAGGCGCCGG AATTCCGATC TGATCAAGAG ACAGGATGAG 
1501 GATCGTTTCG CATGATTGAA CAAGATGGAT TGCACGCAGG TTCTCCGGCC GCTTGGGTGG 
1561 AGAGGCTATT CGGCTATGAC TGGGCACAAC AGACAATCGG CTGCTCTGAT GCCGCCGTGT 
1621 TCCGGCTGTC AGCGCAGGGG CGCCCGGTTC TTTTTGTCAA GACCGACCTG TCCGGTGCCC 
1681 TGAATGAACT GCAGGACGAG GCAGCGCGGC TATCGTGGCT GGCCACGACG GGCGTTCCTT 
1741 GCGCAGCTGT GCTCGACGTT GTCACTGAAG CGGGAAGGGA CTGGCTGCTA TTGGGCGAAG 
1801 TGCCGGGGCA GGATCTCCTG TCATCTCACC TTGCTCCTGC CGAGAAAGTA TCCATCATGG 
1861 CTGATGCAAT GCGGCGGCTG CATACGCTTG ATCCGGCTAC CTGCCCATTC GACCACCAAG 
1921 CGAAACATCG CATCGAGCGA GCACGTACTC GGATGGAAGC CGGTCTTGTC GATCAGGATG 
1981 ATCTGGACGA AGAGCATCAG GGGCTCGCGC CAGCCGAACT GTTCGCCAGG CTCAAGGCGC 
2041 GCATGCCCGA CGGCGAGGAT CTCGTCGTGA CCCATGGCGA TGCCTGCTTG CCGAATATCA 
2101 TGGTGGAAAA TGGCCGCTTT TCTGGATTCA TCGACTGTGG CCGGCTGGGT GTGGCGGACC 
2161 GCTATCAGGA CATAGCGTTG GCTACCCGTG ATATTGCTGA AGAGCTTGGC GGCGAATGGG 
2221 CTGACCGCTT CCTCGTGCTT TACGGTATCG CCGCTCCCGA TTCGCAGCGC ATCGCCTTCT 
2281 ATCGCCTTCT TGACGAGTTC TTCTGAGCGG GACTCTGGGG TTCGAAATGA CCGACCAAGC 
2341 GACGCCCAAC CTGCCATCAC GAGATTTCGA TTCCACCGCC GCCTTCTATG AAAGGTTGGG 
2401 CTTCGGAATC GTTTTCCGGG ACGCCGGCTG GATGATCCTC CAGCGCGGGG ATCTCATGCT 
2461 GGAGTTCTTC GCCCACCCCG GGCTCGATCC CCTCGCGAGT TGGTTCAGCT GCTGCCTGAG 
2521 GCTGGACGAC CTCGCGGAGT TCTACCGGCA GTGCAAATCC GTCGGCATCC AGGAAACCAG 
2581 CAGCGGCTAT CCGCGCATCC ATGCCCCCGA ACTGCAGGAG TGGGGAGGCA CGATGGCCGC 
2641 TTTGGTCGAG GCGGATCCGG CCATTAGCCA TATTATTCAT TGGTTATATA GCATAAATCA 
2701 ATATTGGCTA TTGGCCATTG CATACGTTGT ATCCATATCA TAATATGTAC ATTTATATTG 
2761 GCTCATGTCC AACATTACCG CCATGTTGAC ATTGATTATT GACTAGTTAT TAATAGTAAT 
2821 CAATTACGGG GTCATTAGTT CATAGCCCAT ATATGGAGTT CCGCGTTACA TAACTTACGG 
2881 TAAATGGCCC GCCTGGCTGA CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT 
2941 ATGTTCCCAT AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC 
3001 GGTAAACTGC CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTG 
3061 ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC TTATGGGACT 
3121 TTCCTACTTG GCAGTACATC TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT 
3181 GGCAGTACAT CAATGGGCGT GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC 
3241 CCATTGACGT CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC 
3301 GTAACAACTC CGCCCCATTG ACGCAAATGG GCGGTAGGCA TGTACGGTGG GAGGTCTATA 
3361 TAAGCAGAGC TCGTTTAGTG AACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTG 
3421 ACCTCCATAG AAGACACCGG GACCGATCCA GCCTCCGCGG CCCCAAGCTT CCACCATGTC 
3481 TAGATTAGAT AAAAGTAAAG TGATTAACAG CGCATTAGAG CTGCTTAATG AGGTCGGAAT 
3541 CGAAGGTTTA ACAACCCGTA AACTCGCCCA GAAGCTAGGT GTAGAGCAGC CTACATTGTA 
3601 TTGGCATGTA AAAAATAAGC GGGCTTTGCT CGACGCCTTA GCCATTGAGA TGTTAGATAG 
3661 GCACCATACT CACTTTTGCC CTTTAGAAGG GGAAAGCTGG CAAGATTTTT TACGTAATAA 
3721 CGCTAAAAGT TTTAGATGTG CTTTACTAAG TCATCGCGAT GGAGCAAAAG TACATTTAGG 
3781 TACACGGCCT ACAGAAAAAC AGTATGAAAC TCTCGAAAAT CAATTAGCCT TTTTATGCCA 
3841 ACAAGGTTTT TCACTAGAGA ATGCATTATA TGCACTCAGC GCTGTGGGGC ATTTTACTTT 
3901 AGGTTGCGTA TTGGAAGATC AAGAGCATCA AGTCGCTAAA GAAGAAAGGG AAACACCTAC 
3961 TACTGATAGT ATGCCGCCAT TATTACGACA AGCTATCGAA TTATTTGATC ACCAAGGTGC 
4021 AGAGCCAGCC TTCTTATTCG GCCTTGAATT GATCATATGC GGATTAGAAA AACAACTTAA 
4081 ATGTGAAAGT GGGTCCGCGT ACAGCCGCGC GCGTACGAAA AACAATTACG GGTCTACCAT 
4141 CGAGGGCCTG CTCGATCTCC CGGACGACGA CGCCCCCGAA GAGGCGGGGC TGGCGGCTCC 
4201 GCGCCTGTCC TTTCTCCCCG CGGGACACAC GCGCAGACTG TCGACGGCCC CCCCGACCGA 
4261 TGTCAGCCTG GGGGACGAGC TCCACTTAGA CGGCGAGGAC GTGGCGATGG CGCATGCCGA 
4321 CGCGCTAGAC GATTTCGATC TGGACATGTT GGGGGACGGG GATTCCCCGG GTCCGGGATT 
4381 TACCCCCCAC GACTCCGCCC CCTACGGCGC TCTGGATATG GCCGACTTCG AGTTTGAGCA 
4441 GATGTTTACC GATGCCCTTG GAATTGACGA GTACGGTGGG TAGGGGGCGC GAATCGCGTC 
4501 CGGGGCCGGG GACCTTGCAC AGATAGCGTG GTCCGGCCAG GACGACGAGG CTTTGCAGGA 
4561 TCATAAATCA GCCATACCAC ATTTGTAGAG GTTTTACTTG CTTTAAAAAA ACCTCCCACA 
4621 CCTCCCCCTG AACCTGAAAC ATAAAATGAA TGCAATTGTT GTTGTTCCCG GGGGATCCGT 
4681 TTAAACTTAA TTAAGCGGCC AACATCGATA AAATAAAAGA TTTTATTTAG TCTCCAGAAA 
4741 AAGGGGGGAA TGAAAGACCC CACCTGTAGG TTTGGCAAGC TAGCTTAAGT AACGCCATTT 
4801 TGCAAGGCAT GGAAAAATAC ATAACTGAGA ATAGAGAAGT TCAGATCAAG GTCAGGAACA 
4861 GATGGAACAG CTGAATATGG GCCAAACAGG ATATCTGTGG TAAGCAGTTC CTGCCCCGGC 
4921 TCAGGGCCAA GAACAGATGG AACAGCTGAA TATGGGCCAA ACAGGATATC TGTGGTAAGC 
4981 AGTTCCTGCC CCGGCTCAGG GCCAAGAACA GATGGTCCCC AGATGCGGTC CAGCCCTCAG 
5041 CAGTTTCTAG AGAACCATCA GATGTTTCCA GGGTGCCCCA AGGACCTGAA ATGACCCTGT 
5101 GCCTTATTTG AACTAACCAA TCAGTTCGCT TCTCGCTTCT GTTCGCGCGC TTCTGCTCCC 
5161 CGAGCTCAAT AAAAGAGCCC ACAACCCCTC ACTCGGGGCG CCAGTCCTCC GATTGACTGA 
5221 GTCGCCCGGG TACCCGTGTA TCCAATAAAC CCTCTTGCAG TTGCATCCGA CTTGTGGTCT 
5281 CGCTGTTCCT TGGGAGGGTC TCCTCTGAGT GATTGACTAC CCGTCAGCGG GGGTCTTTCA 
5341 TTTGGGGGCT CGTCCGGGAT CGGGAGACCC CTGCCCAGGG ACCACCGACC CACCACCGGG 
5401 AGGTAAGCTG GCTGCCTCGC GCGTTTCGGT GATGACGGTG AAAACCTCTG ACACATGCAG 
5461 CTCCCGGAGA CGGTCACAGC TTGTCTGTAA GCGGATGCCG GGAGCAGACA AGCCCGTCAG 
5521 GGCGCGTCAG CGGGTGTTGG CGGGTGTCGG GGCGCAGCCA TGACCCAGTC ACGTAGCGAT 
5581 AGCGGAGTGT ATACTGGCTT AACTATGCGG CATCAGAGCA GATTGTACTG AGAGTGCACC 
5641 ATATGCGGTG TGAAATACCG CACAGATGCG TAAGGAGAAA ATACCGCATC AGGCGCTCTT 
5701 CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA GCGGTATCAG 
5761 CTCACTCAAA GGCGGTAATA CGGTTATCCA CAGAATCAGG GGATAACGCA GGAAAGAACA 
5821 TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT 
5881 TCCATAGGCT CCGCCCCCCT GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC 
5941 GAAACCCGAC AGGACTATAA AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT 
6001 CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG 
6061 TGGCGCTTTC TCATAGCTCA CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA 
6121 AGCTGGGCTG TGTGCACGAA CCCCCCGTTC AGCCCGACCG CTGCGCCTTA TCCGGTAACT 
6181 ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA 
6241 ACAGGATTAG CAGAGCGAGG TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA 
6301 ACTACGGCTA CACTAGAAGG ACAGTATTTG GTATCTGCGC TCTGCTGAAG CCAGTTACCT 
6361 TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC CACCGCTGGT AGCGGTGGTT 
6421 TTTTTGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA 
6481 TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA 
6541 TGAGATTATC AAAAAGGATC TTCACCTAGA TCCTTTTAAA TTAAAAATGA AGTTTTAAAT 
6601 CAATCTAAAG TATATATGAG TAAACTTGGT CTGACAGTTA CCAATGCTTA ATCAGTGAGG 
6661 CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT TGCCTGACTC CCCGTCGTGT 
6721 AGATAACTAC GATACGGGAG GGCTTACCAT CTGGCCCCAG TGCTGCAATG ATACCGCGAG 
6781 ACCCACGCTC ACCGGCTCCA GATTTATCAG CAATAAACCA GCCAGCCGGA AGGGCCGAGC 
6841 GCAGAAGTGG TCCTGCAACT TTATCCGCCT CCATCCAGTC TATTAATTGT TGCCGGGAAG 
6901 CTAGAGTAAG TAGTTCGCCA GTTAATAGTT TGCGCAACGT TGTTGCCATT GCTGCAGGCA 
6961 TCGTGGTGTC ACGCTCGTCG TTTGGTATGG CTTCATTCAG CTCCGGTTCC CAACGATCAA 
7021 GGCGAGTTAC ATGATCCCCC ATGTTGTGCA AAAAAGCGGT TAGCTCCTTC GGTCCTCCGA 
7081 TCGTTGTCAG AAGTAAGTTG GCCGCAGTGT TATCACTCAT GGTTATGGCA GCACTGCATA 
7141 ATTCTCTTAC TGTCATGCCA TCCGTAAGAT GCTTTTCTGT GACTGGTGAG TACTCAACCA 
7201 AGTCATTCTG AGAATAGTGT ATGCGGCGAC CGAGTTGCTC TTGCCCGGCG TCAACACGGG 
7261 ATAATACCGC GCCACATAGC AGAACTTTAA AAGTGCTCAT CATTGGAAAA CGTTCTTCGG 
7321 GGCGAAAACT CTCAAGGATC TTACCGCTGT TGAGATCCAG TTCGATGTAA CCCACTCGTG 
7381 CACCCAACTG ATCTTCAGCA TCTTTTACTT TCACCAGCGT TTCTGGGTGA GCAAAAACAG 
7441 GAAGGCAAAA TGCCGCAAAA AAGGGAATAA GGGCGACACG GAAATGTTGA ATACTCATAC 
7501 TCTTCCTTTT TCAATATTAT TGAAGCATTT ATCAGGGTTA TTGTCTCATG AGCGGATACA 
7561 TATTTGAATG TATTTAGAAA AATAAACAAA TAGGGGTTCC GCGCACATTT CCCCGAAAAG 
7621 TGCCACCTGA CGTCTAAGAA ACCATTATTA TCATGACATT AACCTATAAA AATAGGCGTA 
7681 TCACGAGGCC CTTTCGTCTT CAAGAATTCA TACCAGATCA CCGAAAACTG TCCTCCAAAT 
7741 GTGTCCCCCT CACACTCCCA AATTCGCGGG CTTCTGCCTC TTAGACCACT CTACCCTATT 
7801 CCCCACACTC ACCGGAGCCA AAGCCGCGGC CCTTCCGTTT CTTTGCT