pMSCVpuro

The Murine Stem Cell Virus (MSCV) vectors were derived from the Murine Embryonic Stem Cell Virus (MESV) and the LN retroviral vectors (1,2). Upon transfection into a packaging cell line, pMSCVpuro transiently expresses, or integrates and stably expresses, a transcript containing the extended viral packaging signal Ψ+, the puromycin resistance gene, and a gene of interest. The vectors achieve stable, high-level gene expression in hematopoietic and embryonic stem cells through a specifically designed 5' long terminal repeat (LTR). This LTR is from the murine stem cell PCMV virus, and it differs from the MoMuLV LTR used in other retroviral vectors by several point mutations and a deletion. These changes enhance transcriptional activation and prevent transcriptional suppression in embryonic stem and embryonal carcinoma cells. As a result, the LTR drives high-level constitutive expression of a target gene in stem cells or other mammalian cell lines (3). A gene can be cloned into the multiple cloning site immediately downstream of this LTR. The murine phosphoglycerate kinase (PKG) promoter (PPKG) controls expression of the puromycin resistance gene (Puror) for antibiotic selection in eukaryotic cells. pMSCVpuro also contains the pUC origin of replication and E. coli Ampr gene for propagation and antibiotic selection in bacteria.

载体应用

pMSCVpuro can be transfected into a packaging cell line such as the RetroPackTM PT67 Cell Line (Cat. No. 631510). Once in the cell, RNA from the vector is packaged into infectious, replication-incompetent retroviral particles. pMSCVpuro does not contain the gag, pol, and env structural genes necessary for particle formation and replication: these genes are stably integrated into the PT67 genome (4–7). Introduction of pMSCVpuro into a packaging cell line results in production of hightiter, replication-incompetent infectious virus particles. These particles can infect target cells and transmit the gene of interest to them. However, they cannot replicate because the target cells lack viral structural genes. The process of separately introducing and integrating the structural genes into the packaging cell line minimizes the chances of producing replication-competent virus due to recombination events during cell proliferation.

0001 TGAAAGACCC CACCTGTAGG TTTGGCAAGC TAGCTTAAGT AACGCCATTT TGCAAGGCAT 
0061 GGAAAATACA TAACTGAGAA TAGAGAAGTT CAGATCAAGG TTAGGAACAG AGAGACAGCA 
0121 GAATATGGGC CAAACAGGAT ATCTGTGGTA AGCAGTTCCT GCCCCGGCTC AGGGCCAAGA 
0181 ACAGATGGTC CCCAGATGCG GTCCCGCCCT CAGCAGTTTC TAGAGAACCA TCAGATGTTT 
0241 CCAGGGTGCC CCAAGGACCT GAAATGACCC TGTGCCTTAT TTGAACTAAC CAATCAGTTC 
0301 GCTTCTCGCT TCTGTTCGCG CGCTTCTGCT CCCCGAGCTC AATAAAAGAG CCCACAACCC 
0361 CTCACTCGGC GCGCCAGTCC TCCGATAGAC TGCGTCGCCC GGGTACCCGT ATTCCCAATA 
0421 AAGCCTCTTG CTGTTTGCAT CCGAATCGTG GACTCGCTGA TCCTTGGGAG GGTCTCCTCA 
0481 GATTGATTGA CTGCCCACCT CGGGGGTCTT TCATTTGGAG GTTCCACCGA GATTTGGAGA 
0541 CCCCTGCCCA GGGACCACCG ACCCCCCCGC CGGGAGGTAA GCTGGCCAGC GGTCGTTTCG 
0601 TGTCTGTCTC TGTCTTTGTG CGTGTTTGTG CCGGCATCTA ATGTTTGCGC CTGCGTCTGT 
0661 ACTAGTTAGC TAACTAGCTC TGTATCTGGC GGACCCGTGG TGGAACTGAC GAGTTCTGAA 
0721 CACCCGGCCG CAACCCTGGG AGACGTCCCA GGGACTTTGG GGGCCGTTTT TGTGGCCCGA 
0781 CCTGAGGAAG GGAGTCGATG TGGAATCCGA CCCCGTCAGG ATATGTGGTT CTGGTAGGAG 
0841 ACGAGAACCT AAAACAGTTC CCGCCTCCGT CTGAATTTTT GCTTTCGGTT TGGAACCGAA 
0901 GCCGCGCGTC TTGTCTGCTG CAGCGCTGCA GCATCGTTCT GTGTTGTCTC TGTCTGACTG 
0961 TGTTTCTGTA TTTGTCTGAA AATTAGGGCC AGACTGTTAC CACTCCCTTA AGTTTGACCT 
1021 TAGGTCACTG GAAAGATGTC GAGCGGATCG CTCACAACCA GTCGGTAGAT GTCAAGAAGA 
1081 GACGTTGGGT TACCTTCTGC TCTGCAGAAT GGCCAACCTT TAACGTCGGA TGGCCGCGAG 
1141 ACGGCACCTT TAACCGAGAC CTCATCACCC AGGTTAAGAT CAAGGTCTTT TCACCTGGCC 
1201 CGCATGGACA CCCAGACCAG GTCCCCTACA TCGTGACCTG GGAAGCCTTG GCTTTTGACC 
1261 CCCCTCCCTG GGTCAAGCCC TTTGTACACC CTAAGCCTCC GCCTCCTCTT CCTCCATCCG 
1321 CCCCGTCTCT CCCCCTTGAA CCTCCTCGTT CGACCCCGCC TCGATCCTCC CTTTATCCAG 
1381 CCCTCACTCC TTCTCTAGGC GCCGGAATTA GATCTCTCGA GGTTAACGAA TTCTACCGGG 
1441 TAGGGGAGGC GCTTTTCCCA AGGCAGTCTG GAGCATGCGC TTTAGCAGCC CCGCTGGGCA 
1501 CTTGGCGCTA CACAAGTGGC CTCTGGCCTC GCACACATTC CACATCCACC GGTAGGCGCC 
1561 AACCGGCTCC GTTCTTTGGT GGCCCCTTCG CGCCACCTTC TACTCCTCCC CTAGTCAGGA 
1621 AGTTCCCCCC CGCCCCGCAG CTCGCGTCGT GCAGGACGTG ACAAATGGAA GTAGCACGTC 
1681 TCACTAGTCT CGTGCAGATG GACAGCACCG CTGAGCAATG GAAGCGGGTA GGCCTTTGGG 
1741 GCAGCGGCCA ATAGCAGCTT TGCTCCTTCG CTTTCTGGGC TCAGAGGCTG GGAAGGGGTG 
1801 GGTCCGGGGG CGGGCTCAGG GGCGGGCTCA GGGGCGGGGC GGGCGCCCGA AGGTCCTCCG 
1861 GAGGCCCGGC ATTCTGCACG CTTCAAAAGC GCACGTCTGC CGCGCTGTTC TCCTCTTCCT 
1921 CATCTCCGGG CCTTTCGACC TGCAGCCCAA GCTTACCATG ACCGAGTACA AGCCCACGGT 
1981 GCGCCTCGCC ACCCGCGACG ACGTCCCCAG GGCCGTACGC ACCCTCGCCG CCGCGTTCGC 
2041 CGACTACCCC GCCACGCGCC ACACCGTCGA TCCGGACCGC CACATCGAGC GGGTCACCGA 
2101 GCTGCAAGAA CTCTTCCTCA CGCGCGTCGG GCTCGACATC GGCAAGGTGT GGGTCGCGGA 
2161 CGACGGCGCC GCGGTGGCGG TCTGGACCAC GCCGGAGAGC GTCGAAGCGG GGGCGGTGTT 
2221 CGCCGAGATC GGCCCGCGCA TGGCCGAGTT GAGCGGTTCC CGGCTGGCCG CGCAGCAACA 
2281 GATGGAAGGC CTCCTGGCGC CGCACCGGCC CAAGGAGCCC GCGTGGTTCC TGGCCACCGT 
2341 CGGCGTCTCG CCCGACCACC AGGGCAAGGG TCTGGGCAGC GCCGTCGTGC TCCCCGGAGT 
2401 GGAGGCGGCC GAGCGCGCCG GGGTGCCCGC CTTCCTGGAG ACCTCCGCGC CCCGCAACCT 
2461 CCCCTTCTAC GAGCGGCTCG GCTTCACCGT CACCGCCGAC GTCGAGGTGC CCGAAGGACC 
2521 GCGCACCTGG TGCATGACCC GCAAGCCCGG TGCCTGACGC CCGCCCCACG ACCCGCAGCG 
2581 CCCGACCGAA AGGAGCGCAC GACCCCATGC ATCGATAAAA TAAAAGATTT TATTTAGTCT 
2641 CCAGAAAAAG GGGGGAATGA AAGACCCCAC CTGTAGGTTT GGCAAGCTAG CTTAAGTAAC 
2701 GCCATTTTGC AAGGCATGGA AAATACATAA CTGAGAATAG AGAAGTTCAG ATCAAGGTTA 
2761 GGAACAGAGA GACAGCAGAA TATGGGCCAA ACAGGATATC TGTGGTAAGC AGTTCCTGCC 
2821 CCGGCTCAGG GCCAAGAACA GATGGTCCCC AGATGCGGTC CCGCCCTCAG CAGTTTCTAG 
2881 AGAACCATCA GATGTTTCCA GGGTGCCCCA AGGACCTGAA ATGACCCTGT GCCTTATTTG 
2941 AACTAACCAA TCAGTTCGCT TCTCGCTTCT GTTCGCGCGC TTCTGCTCCC CGAGCTCAAT 
3001 AAAAGAGCCC ACAACCCCTC ACTCGGCGCG CCAGTCCTCC GATAGACTGC GTCGCCCGGG 
3061 TACCCGTGTA TCCAATAAAC CCTCTTGCAG TTGCATCCGA CTTGTGGTCT CGCTGTTCCT 
3121 TGGGAGGGTC TCCTCTGAGT GATTGACTAC CCGTCAGCGG GGGTCTTTCA TGGGTAACAG 
3181 TTTCTTGAAG TTGGAGAACA ACATTCTGAG GGTAGGAGTC GAATATTAAG TAATCCTGAC 
3241 TCAATTAGCC ACTGTTTTGA ATCCACATAC TCCAATACTC CTGAAATAGT TCATTATGGA 
3301 CAGCGCAGAA AGAGCTGGGG AGAATTGTGA AATTGTTATC CGCTCACAAT TCCACACAAC 
3361 ATACGAGCCG GAAGCATAAA GTGTAAAGCC TGGGGTGCCT AATGAGTGAG CTAACTCACA 
3421 TTAATTGCGT TGCGCTCACT GCCCGCTTTC CAGTCGGGAA ACCTGTCGTG CCAGCTGCAT 
3481 TAATGAATCG GCCAACGCGC GGGGAGAGGC GGTTTGCGTA TTGGGCGCTC TTCCGCTTCC 
3541 TCGCTCACTG ACTCGCTGCG CTCGGTCGTT CGGCTGCGGC GAGCGGTATC AGCTCACTCA 
3601 AAGGCGGTAA TACGGTTATC CACAGAATCA GGGGATAACG CAGGAAAGAA CATGTGAGCA 
3661 AAAGGCCAGC AAAAGGCCAG GAACCGTAAA AAGGCCGCGT TGCTGGCGTT TTTCCATAGG 
3721 CTCCGCCCCC CTGACGAGCA TCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG 
3781 ACAGGACTAT AAAGATACCA GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG CTCTCCTGTT 
3841 CCGACCCTGC CGCTTACCGG ATACCTGTCC GCCTTTCTCC CTTCGGGAAG CGTGGCGCTT 
3901 TCTCATAGCT CACGCTGTAG GTATCTCAGT TCGGTGTAGG TCGTTCGCTC CAAGCTGGGC 
3961 TGTGTGCACG AACCCCCCGT TCAGCCCGAC CGCTGCGCCT TATCCGGTAA CTATCGTCTT 
4021 GAGTCCAACC CGGTAAGACA CGACTTATCG CCACTGGCAG CAGCCACTGG TAACAGGATT 
4081 AGCAGAGCGA GGTATGTAGG CGGTGCTACA GAGTTCTTGA AGTGGTGGCC TAACTACGGC 
4141 TACACTAGAA GAACAGTATT TGGTATCTGC GCTCTGCTGA AGCCAGTTAC CTTCGGAAAA 
4201 AGAGTTGGTA GCTCTTGATC CGGCAAACAA ACCACCGCTG GTAGCGGTGG TTTTTTTGTT 
4261 TGCAAGCAGC AGATTACGCG CAGAAAAAAA GGATCTCAAG AAGATCCTTT GATCTTTTCT 
4321 ACGGGGTCTG ACGCTCAGTG GAACGAAAAC TCACGTTAAG GGATTTTGGT CATGAGATTA 
4381 TCAAAAAGGA TCTTCACCTA GATCCTTTTA AATTAAAAAT GAAGTTTTAA ATCAATCTAA 
4441 AGTATATATG AGTAAACTTG GTCTGACAGT TACCAATGCT TAATCAGTGA GGCACCTATC 
4501 TCAGCGATCT GTCTATTTCG TTCATCCATA GTTGCCTGAC TCCCCGTCGT GTAGATAACT 
4561 ACGATACGGG AGGGCTTACC ATCTGGCCCC AGTGCTGCAA TGATACCGCG AGACCCACGC 
4621 TCACCGGCTC CAGATTTATC AGCAATAAAC CAGCCAGCCG GAAGGGCCGA GCGCAGAAGT 
4681 GGTCCTGCAA CTTTATCCGC CTCCATCCAG TCTATTAATT GTTGCCGGGA AGCTAGAGTA 
4741 AGTAGTTCGC CAGTTAATAG TTTGCGCAAC GTTGTTGCCA TTGCTACAGG CATCGTGGTG 
4801 TCACGCTCGT CGTTTGGTAT GGCTTCATTC AGCTCCGGTT CCCAACGATC AAGGCGAGTT 
4861 ACATGATCCC CCATGTTGTG CAAAAAAGCG GTTAGCTCCT TCGGTCCTCC GATCGTTGTC 
4921 AGAAGTAAGT TGGCCGCAGT GTTATCACTC ATGGTTATGG CAGCACTGCA TAATTCTCTT 
4981 ACTGTCATGC CATCCGTAAG ATGCTTTTCT GTGACTGGTG AGTACTCAAC CAAGTCATTC 
5041 TGAGAATAGT GTATGCGGCG ACCGAGTTGC TCTTGCCCGG CGTCAATACG GGATAATACC 
5101 GCGCCACATA GCAGAACTTT AAAAGTGCTC ATCATTGGAA AACGTTCTTC GGGGCGAAAA 
5161 CTCTCAAGGA TCTTACCGCT GTTGAGATCC AGTTCGATGT AACCCACTCG TGCACCCAAC 
5221 TGATCTTCAG CATCTTTTAC TTTCACCAGC GTTTCTGGGT GAGCAAAAAC AGGAAGGCAA 
5281 AATGCCGCAA AAAAGGGAAT AAGGGCGACA CGGAAATGTT GAATACTCAT ACTCTTCCTT 
5341 TTTCAATATT ATTGAAGCAT TTATCAGGGT TATTGTCTCA TGAGCGGATA CATATTTGAA 
5401 TGTATTTAGA AAAATAAACA AATAGGGGTT CCGCGCACAT TTCCCCGAAA AGTGCCACCT 
5461 GACGTCTAAG AAACCATTAT TATCATGACA TTAACCTATA AAAATAGGCG TATCACGAGG 
5521 CCCTTTCGTC TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG 
5581 GAGACGGTCA CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG 
5641 TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA 
5701 CTGAGAGTGC ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC 
5761 ATCAGGCGCC ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC 
5821 TCTTCGCTAT TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA 
5881 ACGCCAGGGT TTTCCCAGTC ACGACGTTGT AAAACGACGG CGCAAGGAAT GGTGCATGCA 
5941 AGGAGATGGC GCCCAACAGT CCCCCGGCCA CGGGGCCTGC CACCATACCC ACGCCGAAAC 
6001 AAGCGCTCAT GAGCCCGAAG TGGCGAGCCC GATCTTCCCC ATCGGTGATG TCGGCGATAT 
6061 AGGCGCCAGC AACCGCACCT GTGGCGCCGG TGATGCCGGC CACGATGCGT CCGGCGTAGA 
6121 GGCGATTAGT CCAATTTGTT AAAGACAGGA TATCAGTGGT CCAGGCTCTA GTTTTGACTC 
6181 AACAATATCA CCAGCTGAAG CCTATAGAGT ACGAGCCATA GATAAAATAA AAGATTTTAT 
6241 TTAGTCTCCA GAAAAAGGGG GGAA