pLNCX2

pLNCX2 contains elements derived from Moloney murine leukemia virus (MoMuLV) and Moloney murine sarcoma virus (MoMuSV), and is designed for retroviral gene delivery and expression (1– 3). Upon transfection into a packaging cell line, pLNCX2 can transiently express, or integrate and stably express, a transcript containing Y+ (the extended viral packaging signal) a selectable marker, and the gene of interest. The 5' viral LTR in this vector contains viral promoter/enhancer sequences that control expression of the neomycin resistance (Neor) gene for antibiotic selection in eukaryotic cells. A gene of interest can be cloned into the multiple cloning site immediately downstream of the human cytomegalovirus (CMV) immediate early promoter (PCMV). pLNCX2 also includes the Col E1 origin of replication and E. coli Ampr gene for propagation and antibiotic selection in bacteria.

载体应用

pLNCX2 can be transfected into a packaging cell line such as the RetroPackTM PT67 Cell Line (#K1060-D). Once in the cell, RNA from the vector is packaged into infectious, replicationincompetent retroviral particles. pLNCX2 does not contain the structural genes (gag, pol, and env) necessary for particle formation and replication; these genes are stably integrated into PT67 (4–7). Subsequent introduction of pLNCX2, containing Y+, transcription and processing elements, and the gene of interest produces high-titer, replication-incompetent infectious virus. These retroviral particles can infect target cells and transmit the gene of interest (which is cloned between the viral LTR sequences), but cannot replicate within these cells since the cells lack the 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 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 TTCGAATAGT AATCAATTAC 
2341 GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA CGGTAAATGG 
2401 CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA CGTATGTTCC 
2461 CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT TACGGTAAAC 
2521 TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA TTGACGTCAA 
2581 TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG ACTTTCCTAC 
2641 TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG GTGATGCGGT TTTGGCAGTA 
2701 CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC ACCCCATTGA 
2761 CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT GTCGTAACAA 
2821 CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT ATATAAGCAG 
2881 AGCTGGTTTA GTGAACCGTC AGATCCGCTA GCGCTACCGG ACTCAGATCT CGAGCTCAAG 
2941 CTTGTTTGGC CGAGGCGGCC GCTTGTCGAC AGGCCTTAAT GGCCTAACAT CGATAAAATA 
3001 AAAGATTTTA TTTAGTCTCC AGAAAAAGGG GGGAATGAAA GACCCCACCT GTAGGTTTGG 
3061 CAAGCTAGCT TAAGTAACGC CATTTTGCAA GGCATGGAAA AATACATAAC TGAGAATAGA 
3121 GAAGTTCAGA TCAAGGTCAG GAACAGATGG AACAGCTGAA TATGGGCCAA ACAGGATATC 
3181 TGTGGTAAGC AGTTCCTGCC CCGGCTCAGG GCCAAGAACA GATGGAACAG CTGAATATGG 
3241 GCCAAACAGG ATATCTGTGG TAAGCAGTTC CTGCCCCGGC TCAGGGCCAA GAACAGATGG 
3301 TCCCCAGATG CGGTCCAGCC CTCAGCAGTT TCTAGAGAAC CATCAGATGT TTCCAGGGTG 
3361 CCCCAAGGAC CTGAAATGAC CCTGTGCCTT ATTTGAACTA ACCAATCAGT TCGCTTCTCG 
3421 CTTCTGTTCG CGCGCTTCTG CTCCCCGAGC TCAATAAAAG AGCCCACAAC CCCTCACTCG 
3481 GGGCGCCAGT CCTCCGATTG ACTGAGTCGC CCGGGTACCC GTGTATCCAA TAAACCCTCT 
3541 TGCAGTTGCA TCCGACTTGT GGTCTCGCTG TTCCTTGGGA GGGTCTCCTC TGAGTGATTG 
3601 ACTACCCGTC AGCGGGGGTC TTTCATTTGG GGGCTCGTCC GGGATCGGGA GACCCCTGCC 
3661 CAGGGACCAC CGACCCACCA CCGGGAGGTA AGCTGGCTGC CTCGCGCGTT TCGGTGATGA 
3721 CGGTGAAAAC CTCTGACACA TGCAGCTCCC GGAGACGGTC ACAGCTTGTC TGTAAGCGGA 
3781 TGCCGGGAGC AGACAAGCCC GTCAGGGCGC GTCAGCGGGT GTTGGCGGGT GTCGGGGCGC 
3841 AGCCATGACC CAGTCACGTA GCGATAGCGG AGTGTATACT GGCTTAACTA TGCGGCATCA 
3901 GAGCAGATTG TACTGAGAGT GCACCATATG CGGTGTGAAA TACCGCACAG ATGCGTAAGG 
3961 AGAAAATACC GCATCAGGCG CTCTTCCGCT TCCTCGCTCA CTGACTCGCT GCGCTCGGTC 
4021 GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG TAATACGGTT ATCCACAGAA 
4081 TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC AGCAAAAGGC CAGGAACCGT 
4141 AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA 
4201 AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT 
4261 CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG 
4321 TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA GCTCACGCTG TAGGTATCTC 
4381 AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC 
4441 GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA 
4501 TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT 
4561 ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA GAAGGACAGT ATTTGGTATC 
4621 TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA 
4681 CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA 
4741 AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA 
4801 AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTT 
4861 TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC 
4921 AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC 
4981 ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC 
5041 CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT ATCAGCAATA 
5101 AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG CAACTTTATC CGCCTCCATC 
5161 CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC 
5221 AACGTTGTTG CCATTGCTGC AGGCATCGTG GTGTCACGCT CGTCGTTTGG TATGGCTTCA 
5281 TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA 
5341 GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGC AGTGTTATCA 
5401 CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA TGCCATCCGT AAGATGCTTT 
5461 TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT 
5521 TGCTCTTGCC CGGCGTCAAC ACGGGATAAT ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG 
5581 CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA 
5641 TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC 
5701 AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG AATAAGGGCG 
5761 ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG 
5821 GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG 
5881 GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTCT AAGAAACCAT TATTATCATG 
5941 ACATTAACCT ATAAAAATAG GCGTATCACG AGGCCCTTTC GTCTTCAAGA ATTCATACCA 
6001 GATCACCGAA AACTGTCCTC CAAATGTGTC CCCCTCACAC TCCCAAATTC GCGGGCTTCT 
6061 GCCTCTTAGA CCACTCTACC CTATTCCCCA CACTCACCGG AGCCAAAGCC GCGGCCCTTC 
6121 CGTTTCTTTG CT