pQCXIH Retroviral Vector is a self-inactivating bicistronic expression vector designed to express a target gene along with the hygromycin resistance gene (1). Upon transfection into a packaging cell line, this vector can transiently express, or integrate and stably express, a viral genomic transcript containing the CMV immediate early promoter, gene of interest, IRES and the hygromycin resistance gene (Hygr). The gene of interest and the hygromycin resistance gene are co-translated, via the internal ribosome entry site (IRES), as a bicistronic message in mammalian cells (2, 3).

This vector incorporates unique features including: optimization to remove promoter interference and self-inactivation. The hybrid 5' LTR consists of the cytomegalovirus (CMV) type I enhancer and the mouse sarcoma virus (MSV) promoter. This construct drives high levels of transcription in HEK 293-based packaging cell lines due, in part, to the presence of adenoviral E1A (4, 5, 6, 7) in these cells. The self-inactivating feature of the vector is provided by a deletion in the 3' LTR enhancer region (U3). During reverse transcription of the retroviral RNA, the inactivated 3' LTR is copied and replaces the 5' LTR, resulting in inactivation of the 5' LTR CMV enhancer sequences. This may reduce the phenomenon known as promoter interference (8) and allow more efficient expression.

Also included in the viral genomic transcript are the necessary viral RNA processing elements including the LTRs, packaging signal (Psi+), and tRNA primer binding site. pQCXIH also contains a bacterial origin of replication and E. coli Ampr gene for propagation and selection in bacteria.

載體應(yīng)用

pQCXIH is designed to deliver and express a gene of interest, along with the hygromycin resistance marker, from a bicistronic message. The design is optimized to produce high titers via the PCMV IE in the packaging cell line. The bicistronic transcript makes it unnecessary to screen the transformants since the Hygromycin resistance is expressed in concert with the gene inserted into the multiple cloning site.

Once transfected into the packaging cell line (such as the RetroPack? PT67 Cell Line; Cat. No. 631510), RNA from the vector is packaged into infectious, replication-incompetent retroviral particles since pQCXIH lacks structural genes (gag, pol, and env) necessary for particle formation and replication; however, these genes are stably integrated as part of the packaging cell genome. Once a high titer clone is selected, these retroviral particles can infect target cells and transmit the gene of interest but cannot replicate within these cells due to the absence of viral structural genes. The separate introduction and integration of the structural genes into the packaging cell line minimizes the chances of producing replication-competent virus due to recombination events during cell proliferation.

0001 TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC 
0061 CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC 
0121 GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA 
0181 TGCCAAGTAC GCCCCCTATT GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC 
0241 AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT CTACGTATTA GTCATCGCTA 
0301 TTACCATGGT GATGCGGTTT TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC 
0361 GGGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGG CACCAAAATC 
0421 AACGGGACTT TCCAAAATGT CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC 
0481 GTGTACGGTG GGAGGTCTAT ATAAGCAGAG CTCAATAAAA GAGCCCACAA CCCCTCACTC 
0541 GGCGCGCCAG TCTTCCGATA GACTGCGTCG CCCGGGTACC CGTATTCCCA ATAAAGCCTC 
0601 TTGCTGTTTG CATCCGAATC GTGGTCTCGC TGTTCCTTGG GAGGGTCTCC TCTGAGTGAT 
0661 TGACTACCCA CGACGGGGGT CTTTCATTTG GGGGCTCGTC CGGGATTTGG AGACCCCTGC 
0721 CCAGGGACCA CCGACCCACC ACCGGGAGGT AAGCTGGCCA GCAACTTATC TGTGTCTGTC 
0781 CGATTGTCTA GTGTCTATGT TTGATGTTAT GCGCCTGCGT CTGTACTAGT TAGCTAACTA 
0841 GCTCTGTATC TGGCGGACCC GTGGTGGAAC TGACGAGTTC TGAACACCCG GCCGCAACCC 
0901 TGGGAGACGT CCCAGGGACT TTGGGGGCCG TTTTTGTGGC CCGACCTGAG GAAGGGAGTC 
0961 GATGTGGAAT CCGACCCCGT CAGGATATGT GGTTCTGGTA GGAGACGAGA ACCTAAAACA 
1021 GTTCCCGCCT CCGTCTGAAT TTTTGCTTTC GGTTTGGAAC CGAAGCCGCG CGTCTTGTCT 
1081 GCTGCAGCGC TGCAGCATCG TTCTGTGTTG TCTCTGTCTG ACTGTGTTTC TGTATTTGTC 
1141 TGAAAATTAG GGCCAGACTG TTACCACTCC CTTAAGTTTG ACCTTAGGTC ACTGGAAAGA 
1201 TGTCGAGCGG ATCGCTCACA ACCAGTCGGT AGATGTCAAG AAGAGACGTT GGGTTACCTT 
1261 CTGCTCTGCA GAATGGCCAA CCTTTAACGT CGGATGGCCG CGAGACGGCA CCTTTAACCG 
1321 AGACCTCATC ACCCAGGTTA AGATCAAGGT CTTTTCACCT GGCCCGCATG GACACCCAGA 
1381 CCAGGTCCCC TACATCGTGA CCTGGGAAGC CTTGGCTTTT GACCCCCCTC CCTGGGTCAA 
1441 GCCCTTTGTA CACCCTAAGC CTCCGCCTCC TCTTCCTCCA TCCGCCCCGT CTCTCCCCCT 
1501 TGAACCTCCT CGTTCGACCC CGCCTCGATC CTCCCTTTAT CCAGCCCTCA CTCCTTCTCT 
1561 AGGCGCCGGA ATTGAAGATC TGGGGGATCG ATCCTCTAGA GTCCGTTACA TAACTTACGG 
1621 TAAATGGCCC GCCTGGCTGA CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT 
1681 ATGTTCCCAT AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC 
1741 GGTAAACTGC CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTG 
1801 ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC TTATGGGACT 
1861 TTCCTACTTG GCAGTACATC TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT 
1921 GGCAGTACAT CAATGGGCGT GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC 
1981 CCATTGACGT CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC 
2041 GTAACAACTC CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA 
2101 TAAGCAGAGC TCGTTTAGTG AACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTG 
2161 ACCTCCATAG AAGACACCGG GACCGATCCA GCCTCCGCGG CCGGGAACGG TGCATTGGAA 
2221 CGGACCTGCA GGAATTGATC CGCGGCCGCA CCGGTAGGCC TCGTACGCTT AATTAACGGA 
2281 TCCGGAATTC CGCCCCCCCC CCCCCCCCCC CTCTCCCTCC CCCCCCCCTA ACGTTACTGG 
2341 CCGAAGCCGC TTGGAATAAG GCCGGTGTGC GTTTGTCTAT ATGTTATTTT CCACCATATT 
2401 GCCGTCTTTT GGCAATGTGA GGGCCCGGAA ACCTGGCCCT GTCTTCTTGA CGAGCATTCC 
2461 TAGGGGTCTT TCCCCTCTCG CCAAAGGAAT GCAAGGTCTG TTGAATGTCG TGAAGGAAGC 
2521 AGTTCCTCTG GAAGCTTCTT GAAGACAAAC AACGTCTGTA GCGACCCTTT GCAGGCAGCG 
2581 GAACCCCCCA CCTGGCGACA GGTGCCTCTG CGGCCAAAAG CCACGTGTAT AAGATACACC 
2641 TGCAAAGGCG GCACAACCCC AGTGCCACGT TGTGAGTTGG ATAGTTGTGG AAAGAGTCAA 
2701 ATGGCTCTCC TCAAGCGTAT TCAACAAGGG GCTGAAGGAT GCCCAGAAGG TACCCCATTG 
2761 TATGGGATCT GATCTGGGGC CTCGGTGCAC ATGCTTTACA TGTGTTTAGT CGAGGTTAAA 
2821 AAAACGTCTA GGCCCCCCGA ACCACGGGGA CGTGGTTTTC CTTTGAAAAA CACGATGATA 
2881 AGCTTGCCAC AACCCGTACC AAAGATGGAT AGATCCGGAA AGCCTGAACT CACCGCGACG 
2941 TCTGTCGAGA AGTTTCTGAT CGAAAAGTTC GACAGCGTCT CCGACCTGAT GCAGCTCTCG 
3001 GAGGGCGAAG AATCTCGTGC TTTCAGCTTC GATGTAGGAG GGCGTGGATA TGTCCTGCGG 
3061 GTAAATAGCT GCGCCGATGG TTTCTACAAA GATCGTTATG TTTATCGGCA CTTTGCATCG 
3121 GCCGCGCTCC CGATTCCGGA AGTGCTTGAC ATTGGGGAAT TCAGCGAGAG CCTGACCTAT 
3181 TGCATCTCCC GCCGTGCACA GGGTGTCACG TTGCAAGACC TGCCTGAAAC CGAACTGCCC 
3241 GCTGTTCTGC AGCCGGTCGC GGAGGCCATG GATGCGATCG CTGCGGCCGA TCTTAGCCAG 
3301 ACGAGCGGGT TCGGCCCATT CGGACCGCAA GGAATCGGTC AATACACTAC ATGGCGTGAT 
3361 TTCATATGCG CGATTGCTGA TCCCCATGTG TATCACTGGC AAACTGTGAT GGACGACACC 
3421 GTCAGTGCGT CCGTCGCGCA GGCTCTCGAT GAGCTGATGC TTTGGGCCGA GGACTGCCCC 
3481 GAAGTCCGGC ACCTCGTGCA CGCGGATTTC GGCTCCAACA ATGTCCTGAC GGACAATGGC 
3541 CGCATAACAG CGGTCATTGA CTGGAGCGAG GCGATGTTCG GGGATTCCCA ATACGAGGTC 
3601 GCCAACATCT TCTTCTGGAG GCCGTGGTTG GCTTGTATGG AGCAGCAGAC GCGCTACTTC 
3661 GAGCGGAGGC ATCCGGAGCT TGCAGGATCG CCGCGGCTCC GGGCGTATAT GCTCCGCATT 
3721 GGTCTTGACC AACTCTATCA GAGCTTGGTT GACGGCAATT TCGATGATGC AGCTTGGGCG 
3781 CAGGGTCGAT GCGACGCAAT CGTCCGATCC GGAGCCGGGA CTGTCGGGCG TACACAAATC 
3841 GCCCGCAGAA GCGCGGCCGT CTGGACCGAT GGCTGTGTAG AAGTACTCGC CGATAGTGGA 
3901 AACCGACGCC CCAGCACTCG TCCGAGGGCA AAGGAATAGA GTAGATGCCG ACCGAACAAG 
3961 AGCTGATTTC GAGAACGCCT CAGCCAGCAA CTCGCGCGAG CCTAGCAAGG CAAATGCGAG 
4021 AGAACGGCCT TACGCTTGGT GGCACAGTTC TCGTCCACAG TTCGCTAAGC TCGCTCGGCT 
4081 GGGTCGCGGG AGGGCCGGTC GCAGTGATTC AGGCCCTTCT GGATTGTGTT GGTCCCCAGG 
4141 GCACGATTGT CATGCCCACG CACTCGGGTG ATCTGACTGA TCCCGCAGAT TGGAGATCGC 
4201 CGCCCGTGCC TGCCGATTGG GTGCAGATCT CTATCGAGGG CTGCAGCGCT GCAGAGGCCG 
4261 AGTGCAGAAC TGCTCCAAAG GGACCTCAAG GCTTTCCGAG GGACACTAGG CTGACTCCAT 
4321 CGAGCCAGTG TAGAGATAAG CTTATCGATT AGTCCAATTT GTTAAAGACA GGATATCAGT 
4381 GGTCCAGGCT CTAGTTTTGA CTCAACAATA TCACCAGCTG AAGCCTATAG AGTACGAGCC 
4441 ATAGATAAAA TAAAAGATTT TATTTAGTCT CCAGAAAAAG GGGGGAATGA AAGACCCCAC 
4501 CTGTAGGTTT GGCAAGCTAG CTTAAGTAAC GCCATTTTGC AAGGCATGGA AAAATACATA 
4561 ACTGAGAATA GAGAAGTTCA GATCAAGGTC AGGAACAGAT GGAACAGGGT CGACCCTAGA 
4621 GAACCATCAG ATGTTTCCAG GGTGCCCCAA GGACCTGAAA TGACCCTGTG CCTTATTTGA 
4681 ACTAACCAAT CAGTTCGCTT CTCGCTTCTG TTCGCGCGCT TCTGCTCCCC GAGCTCAATA 
4741 AAAGAGCCCA CAACCCCTCA CTCGGGGCGC CAGTCCTCCG ATTGACTGAG TCGCCCGGGT 
4801 ACCCGTGTAT CCAATAAACC CTCTTGCAGT TGCATCCGAC TTGTGGTCTC GCTGTTCCTT 
4861 GGGAGGGTCT CCTCTGAGTG ATTGACTACC CGTCAGCGGG GGTCTTTCAT TTGGGGGCTC 
4921 GTCCGGGATC GGGAGACCCC TGCCCAGGGA CCACCGACCC ACCACCGGGA GGTAAGCTGG 
4981 CTGCCTCGCG CGTTTCGGTG ATGACGGTGA AAACCTCTGA CACATGCAGC TCCCGGAGAC 
5041 GGTCACAGCT TGTCTGTAAG CGGATGCCGG GAGCAGACAA GCCCGTCAGG GCGCGTCAGC 
5101 GGGTGTTGGC GGGTGTCGGG GCGCAGCCAT GACCCAGTCA CGTAGCGATA GCGGAGTGTA 
5161 GATCCGGCTG TGGAATGTGT GTCAGTTAGG GTGTGGAAAG TCCCCAGGCT CCCCAGCAGG 
5221 CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC AGGTGTGGAA AGTCCCCAGG 
5281 CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCATAGTCCC 
5341 GCCCCTAACT CCGCCCATCC CGCCCCTAAC TCCGCCCAGT TCCGCCCATT CTCCGCCCCA 
5401 TGGCTGACTA ATTTTTTTTA TTTATGCAGA GGCCGAGGCC GCCTCGGCCT CTGAGCTATT 
5461 CCAGAAGTAG TGAGGAGGCT TTTTTGGAGG CCTAGGCTTT TGCAAAAAGC TTACTGGCTT 
5521 AACTATGCGG CATCAGAGCA GATTGTACTG AGAGTGCACC ATATGCGGTG TGAAATACCG 
5581 CACAGATGCG TAAGGAGAAA ATACCGCATC AGGCGCTCTT CCGCTTCCTC GCTCACTGAC 
5641 TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA GCGGTATCAG CTCACTCAAA GGCGGTAATA 
5701 CGGTTATCCA CAGAATCAGG GGATAACGCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA 
5761 AAGGCCAGGA ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT 
5821 GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC GAAACCCGAC AGGACTATAA 
5881 AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG 
5941 CTTACCGGAT ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC TCATAGCTCA 
6001 CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA 
6061 CCCCCCGTTC AGCCCGACCG CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG 
6121 GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA ACAGGATTAG CAGAGCGAGG 
6181 TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA ACTACGGCTA CACTAGAAGG 
6241 ACAGTATTTG GTATCTGCGC TCTGCTGAAG CCAGTTACCT TCGGAAAAAG AGTTGGTAGC 
6301 TCTTGATCCG GCAAACAAAC CACCGCTGGT AGCGGTGGTT TTTTTGTTTG CAAGCAGCAG 
6361 ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA TCTTTTCTAC GGGGTCTGAC 
6421 GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC 
6481 TTCACCTAGA TCCTTTTAAA TTAAAAATGA AGTTTTAAAT CAATCTAAAG TATATATGAG 
6541 TAAACTTGGT CTGACAGTTA CCAATGCTTA ATCAGTGAGG CACCTATCTC AGCGATCTGT 
6601 CTATTTCGTT CATCCATAGT TGCCTGACTC CCCGTCGTGT AGATAACTAC GATACGGGAG 
6661 GGCTTACCAT CTGGCCCCAG TGCTGCAATG ATACCGCGAG ACCCACGCTC ACCGGCTCCA 
6721 GATTTATCAG CAATAAACCA GCCAGCCGGA AGGGCCGAGC GCAGAAGTGG TCCTGCAACT 
6781 TTATCCGCCT CCATCCAGTC TATTAATTGT TGCCGGGAAG CTAGAGTAAG TAGTTCGCCA 
6841 GTTAATAGTT TGCGCAACGT TGTTGCCATT GCTGCAGGCA TCGTGGTGTC ACGCTCGTCG 
6901 TTTGGTATGG CTTCATTCAG CTCCGGTTCC CAACGATCAA GGCGAGTTAC ATGATCCCCC 
6961 ATGTTGTGCA AAAAAGCGGT TAGCTCCTTC GGTCCTCCGA TCGTTGTCAG AAGTAAGTTG 
7021 GCCGCAGTGT TATCACTCAT GGTTATGGCA GCACTGCATA ATTCTCTTAC TGTCATGCCA 
7081 TCCGTAAGAT GCTTTTCTGT GACTGGTGAG TACTCAACCA AGTCATTCTG AGAATAGTGT 
7141 ATGCGGCGAC CGAGTTGCTC TTGCCCGGCG TCAACACGGG ATAATACCGC GCCACATAGC 
7201 AGAACTTTAA AAGTGCTCAT CATTGGAAAA CGTTCTTCGG GGCGAAAACT CTCAAGGATC 
7261 TTACCGCTGT TGAGATCCAG TTCGATGTAA CCCACTCGTG CACCCAACTG ATCTTCAGCA 
7321 TCTTTTACTT TCACCAGCGT TTCTGGGTGA GCAAAAACAG GAAGGCAAAA TGCCGCAAAA 
7381 AAGGGAATAA GGGCGACACG GAAATGTTGA ATACTCATAC TCTTCCTTTT TCAATATTAT 
7441 TGAAGCATTT ATCAGGGTTA TTGTCTCATG AGCGGATACA TATTTGAATG TATTTAGAAA 
7501 AATAAACAAA TAGGGGTTCC GCGCACATTT CCCCGAAAAG TGCCACCTGA CGTCTAAGAA 
7561 ACCATTATTA TCATGACATT AACCTATAAA AATAGGCGTA TCACGAGGCC CTTTCGTCTT 
7621 CAAGAATTAG CTTGGCCATT GCATACGTTG TATCCATATC ATAATATGTA CATTTATATT 
7681 GGCTCATGTC CAACATTACC GCCATGTTGA CATTGATTAT TGACTAGTTA TTAATAGTAA 
7741 TCAATTACGG GGTCATTAGT TCATAGCCCA TATATGGAGT