pRevTRE

pRevTRE is a retroviral Tet response vector that expresses a gene of interest from the Tetresponse element (TRE). 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). The TRE contains seven direct repeats of the tetO operator sequence, upstream of a minimal CMV promoter, which can be bound by the tTA and rtTA transactivators. The 5' viral LTR controls expression of the transcript that contains Ψ+ (the extended viral packaging signal) and the hygromycin resistance (Hygr) gene for antibiotic selection in mammalian cells. The TRE is derived from vectors described previously (2, 3). pRevTRE also includes the E. coli Ampr gene for antibiotic selection in bacteria.

The complete RevTet-Off and RevTet-On Systems also include the control vector pRevTRELuc, which was constructed by cloning the firefly luciferase gene into the Hind III/Cla I sites in the MCS of pRevTRE.

载体应用

pRevTRE can be used to establish inducible Tet Systems via retrovirus-mediated gene transfer (4). Retroviral gene transfer allows the highly efficient transduction of virtually all dividing cell types. The RevTetTM Systems are also suitable for establishing transgenic animals. In combination with the pRevTet-On or pRevTet- Off regulatory 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 and rtTA bind to the Tetresponse element (TRE) and activate transcription from the minimal promoter in the absence or presence of Dox, respectively. pRevTRE 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 pRevTRE 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 TGATAGCTTG CCACAACCCG 
1501 TACCAAAGAT GGATAGATCC GGAAAGCCTG AACTCACCGC GACGTCTGTC GAGAAGTTTC 
1561 TGATCGAAAA GTTCGACAGC GTCTCCGACC TGATGCAGCT CTCGGAGGGC GAAGAATCTC 
1621 GTGCTTTCAG CTTCGATGTA GGAGGGCGTG GATATGTCCT GCGGGTAAAT AGCTGCGCCG 
1681 ATGGTTTCTA CAAAGATCGT TATGTTTATC GGCACTTTGC ATCGGCCGCG CTCCCGATTC 
1741 CGGAAGTGCT TGACATTGGG GAATTCAGCG AGAGCCTGAC CTATTGCATC TCCCGCCGTG 
1801 CACAGGGTGT CACGTTGCAA GACCTGCCTG AAACCGAACT GCCCGCTGTT CTGCAGCCGG 
1861 TCGCGGAGGC CATGGATGCG ATCGCTGCGG CCGATCTTAG CCAGACGAGC GGGTTCGGCC 
1921 CATTCGGACC GCAAGGAATC GGTCAATACA CTACATGGCG TGATTTCATA TGCGCGATTG 
1981 CTGATCCCCA TGTGTATCAC TGGCAAACTG TGATGGACGA CACCGTCAGT GCGTCCGTCG 
2041 CGCAGGCTCT CGATGAGCTG ATGCTTTGGG CCGAGGACTG CCCCGAAGTC CGGCACCTCG 
2101 TGCACGCGGA TTTCGGCTCC AACAATGTCC TGACGGACAA TGGCCGCATA ACAGCGGTCA 
2161 TTGACTGGAG CGAGGCGATG TTCGGGGATT CCCAATACGA GGTCGCCAAC ATCTTCTTCT 
2221 GGAGGCCGTG GTTGGCTTGT ATGGAGCAGC AGACGCGCTA CTTCGAGCGG AGGCATCCGG 
2281 AGCTTGCAGG ATCGCCGCGG CTCCGGGCGT ATATGCTCCG CATTGGTCTT GACCAACTCT 
2341 ATCAGAGCTT GGTTGACGGC AATTTCGATG ATGCAGCTTG GGCGCAGGGT CGATGCGACG 
2401 CAATCGTCCG ATCCGGAGCC GGGACTGTCG GGCGTACACA AATCGCCCGC AGAAGCGCGG 
2461 CCGTCTGGAC CGATGGCTGT GTAGAAGTAC TCGCCGATAG TGGAAACCGA CGCCCCAGCA 
2521 CTCGTCCGAG GGCAAAGGAA TAGAGTAGAT GCCGACCGAA CAAGAGCTGA TTTCGAGAAC 
2581 GCCTCAGCCA GCAACTCGCG CGAGCCTAGC AAGGCAAATG CGAGAGAACG GCCTTACGCT 
2641 TGGTGGCACA GTTCTCGTCC ACAGTTCGCT AAGCTCGCTC GGCTGGGTCG CGGGAGGGCC 
2701 GGTCGCAGTG ATTCAGGCCC TTCTGGATTG TGTTGGTCCC CAGGGCACGA TTGTCATGCC 
2761 CACGCACTCG GGTGATCTGA CTGATCCCGC AGATTGGAGA TCGCCGCCCG TGCCTGCCGA 
2821 TTGGGTGCAG ATCTCGAGTT TACCACTCCC TATCAGTGAT AGAGAAAAGT GAAAGTCGAG 
2881 TTTACCACTC CCTATCAGTG ATAGAGAAAA GTGAAAGTCG AGTTTACCAC TCCCTATCAG 
2941 TGATAGAGAA AAGTGAAAGT CGAGTTTACC ACTCCCTATC AGTGATAGAG AAAAGTGAAA 
3001 GTCGAGTTTA CCACTCCCTA TCAGTGATAG AGAAAAGTGA AAGTCGAGTT TACCACTCCC 
3061 TATCAGTGAT AGAGAAAAGT GAAAGTCGAG TTTACCACTC CCTATCAGTG ATAGAGAAAA 
3121 GTGAAAGTCG AGCTCGGTAC CCGGGTCGAG TAGGCGTGTA CGGTGGGAGG CCTATATAAG 
3181 CAGAGCTCGT TTAGTGAACC GTCAGATCGC CTGGAGACGC CATCCACGCT GTTTTGACCT 
3241 CCATAGAAGA CACCGGGACC GATCCAGCCT CCGCGGCCCC GAATTCGAGC TCGGTACCCG 
3301 GGGATCCTCT AGAGTCGACC TGCAGGCATG CAAGCTTGTT AACATCGATA AAATAAAAGA 
3361 TTTTATTTAG TCTCCAGAAA AAGGGGGGAA TGAAAGACCC CACCTGTAGG TTTGGCAAGC 
3421 TAGCTTAAGT AACGCCATTT TGCAAGGCAT GGAAAAATAC ATAACTGAGA ATAGAGAAGT 
3481 TCAGATCAAG GTCAGGAACA GATGGAACAG CTGAATATGG GCCAAACAGG ATATCTGTGG 
3541 TAAGCAGTTC CTGCCCCGGC TCAGGGCCAA GAACAGATGG AACAGCTGAA TATGGGCCAA 
3601 ACAGGATATC TGTGGTAAGC AGTTCCTGCC CCGGCTCAGG GCCAAGAACA GATGGTCCCC 
3661 AGATGCGGTC CAGCCCTCAG CAGTTTCTAG AGAACCATCA GATGTTTCCA GGGTGCCCCA 
3721 AGGACCTGAA ATGACCCTGT GCCTTATTTG AACTAACCAA TCAGTTCGCT TCTCGCTTCT 
3781 GTTCGCGCGC TTCTGCTCCC CGAGCTCAAT AAAAGAGCCC ACAACCCCTC ACTCGGGGCG 
3841 CCAGTCCTCC GATTGACTGA GTCGCCCGGG TACCCGTGTA TCCAATAAAC CCTCTTGCAG 
3901 TTGCATCCGA CTTGTGGTCT CGCTGTTCCT TGGGAGGGTC TCCTCTGAGT GATTGACTAC 
3961 CCGTCAGCGG GGGTCTTTCA TTTGGGGGCT CGTCCGGGAT CGGGAGACCC CTGCCCAGGG 
4021 ACCACCGACC CACCACCGGG AGGTAAGCTG GCTGCCTCGC GCGTTTCGGT GATGACGGTG 
4081 AAAACCTCTG ACACATGCAG CTCCCGGAGA CGGTCACAGC TTGTCTGTAA GCGGATGCCG 
4141 GGAGCAGACA AGCCCGTCAG GGCGCGTCAG CGGGTGTTGG CGGGTGTCGG GGCGCAGCCA 
4201 TGACCCAGTC ACGTAGCGAT AGCGGAGTGT ATACTGGCTT AACTATGCGG CATCAGAGCA 
4261 GATTGTACTG AGAGTGCACC ATATGCGGTG TGAAATACCG CACAGATGCG TAAGGAGAAA 
4321 ATACCGCATC AGGCGCTCTT CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG 
4381 GCTGCGGCGA GCGGTATCAG CTCACTCAAA GGCGGTAATA CGGTTATCCA CAGAATCAGG 
4441 GGATAACGCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA 
4501 GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT GACGAGCATC ACAAAAATCG 
4561 ACGCTCAAGT CAGAGGTGGC GAAACCCGAC AGGACTATAA AGATACCAGG CGTTTCCCCC 
4621 TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC 
4681 CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC TCATAGCTCA CGCTGTAGGT ATCTCAGTTC 
4741 GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CCCCCCGTTC AGCCCGACCG 
4801 CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC 
4861 ACTGGCAGCA GCCACTGGTA ACAGGATTAG CAGAGCGAGG TATGTAGGCG GTGCTACAGA 
4921 GTTCTTGAAG TGGTGGCCTA ACTACGGCTA CACTAGAAGG ACAGTATTTG GTATCTGCGC 
4981 TCTGCTGAAG CCAGTTACCT TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC 
5041 CACCGCTGGT AGCGGTGGTT TTTTTGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG 
5101 ATCTCAAGAA GATCCTTTGA TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC 
5161 ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC TTCACCTAGA TCCTTTTAAA 
5221 TTAAAAATGA AGTTTTAAAT CAATCTAAAG TATATATGAG TAAACTTGGT CTGACAGTTA 
5281 CCAATGCTTA ATCAGTGAGG CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT 
5341 TGCCTGACTC CCCGTCGTGT AGATAACTAC GATACGGGAG GGCTTACCAT CTGGCCCCAG 
5401 TGCTGCAATG ATACCGCGAG ACCCACGCTC ACCGGCTCCA GATTTATCAG CAATAAACCA 
5461 GCCAGCCGGA AGGGCCGAGC GCAGAAGTGG TCCTGCAACT TTATCCGCCT CCATCCAGTC 
5521 TATTAATTGT TGCCGGGAAG CTAGAGTAAG TAGTTCGCCA GTTAATAGTT TGCGCAACGT 
5581 TGTTGCCATT GCTGCAGGCA TCGTGGTGTC ACGCTCGTCG TTTGGTATGG CTTCATTCAG 
5641 CTCCGGTTCC CAACGATCAA GGCGAGTTAC ATGATCCCCC ATGTTGTGCA AAAAAGCGGT 
5701 TAGCTCCTTC GGTCCTCCGA TCGTTGTCAG AAGTAAGTTG GCCGCAGTGT TATCACTCAT 
5761 GGTTATGGCA GCACTGCATA ATTCTCTTAC TGTCATGCCA TCCGTAAGAT GCTTTTCTGT 
5821 GACTGGTGAG TACTCAACCA AGTCATTCTG AGAATAGTGT ATGCGGCGAC CGAGTTGCTC 
5881 TTGCCCGGCG TCAACACGGG ATAATACCGC GCCACATAGC AGAACTTTAA AAGTGCTCAT 
5941 CATTGGAAAA CGTTCTTCGG GGCGAAAACT CTCAAGGATC TTACCGCTGT TGAGATCCAG 
6001 TTCGATGTAA CCCACTCGTG CACCCAACTG ATCTTCAGCA TCTTTTACTT TCACCAGCGT 
6061 TTCTGGGTGA GCAAAAACAG GAAGGCAAAA TGCCGCAAAA AAGGGAATAA GGGCGACACG 
6121 GAAATGTTGA ATACTCATAC TCTTCCTTTT TCAATATTAT TGAAGCATTT ATCAGGGTTA 
6181 TTGTCTCATG AGCGGATACA TATTTGAATG TATTTAGAAA AATAAACAAA TAGGGGTTCC 
6241 GCGCACATTT CCCCGAAAAG TGCCACCTGA CGTCTAAGAA ACCATTATTA TCATGACATT 
6301 AACCTATAAA AATAGGCGTA TCACGAGGCC CTTTCGTCTT CAAGAATTCA TACCAGATCA 
6361 CCGAAAACTG TCCTCCAAAT GTGTCCCCCT CACACTCCCA AATTCGCGGG CTTCTGCCTC 
6421 TTAGACCACT CTACCCTATT CCCCACACTC ACCGGAGCCA AAGCCGCGGC CCTTCCGTTT 
6481 CTTTGCT