pMSCVneo

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, pMSCVneo transiently expresses, or integrates and stably expresses, a transcript containing the extended viral packaging signal Ψ+, the neomycin 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 neomycin resistance gene (Neor) for antibiotic selection in eukaryotic cells. pMSCVneo also contains the pUC origin of replication and E. coli Ampr gene for propagation and antibiotic selection in bacteria.

载体应用

pMSCVneo 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, replicationincompetent retroviral particles. pMSCVneo 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 pMSCVneo 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 GCCGGAATTC GTTAACCTCG AGAGATCTAA 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 TGCAGCCAAT ATGGGATCGG CCATTGAACA AGATGGATTG 
1981 CACGCAGGTT CTCCGGCCGC TTGGGTGGAG AGGCTATTCG GCTATGACTG GGCACAACAG 
2041 ACAATCGGCT GCTCTGATGC CGCCGTGTTC CGGCTGTCAG CGCAGGGGCG CCCGGTTCTT 
2101 TTTGTCAAGA CCGACCTGTC CGGTGCCCTG AATGAACTGC AGGACGAGGC AGCGCGGCTA 
2161 TCGTGGCTGG CCACGACGGG CGTTCCTTGC GCAGCTGTGC TCGACGTTGT CACTGAAGCG 
2221 GGAAGGGACT GGCTGCTATT GGGCGAAGTG CCGGGGCAGG ATCTCCTGTC ATCTCACCTT 
2281 GCTCCTGCCG AGAAAGTATC CATCATGGCT GATGCAATGC GGCGGCTGCA TACGCTTGAT 
2341 CCGGCTACCT GCCCATTCGA CCACCAAGCG AAACATCGCA TCGAGCGAGC ACGTACTCGG 
2401 ATGGAAGCCG GTCTTGTCGA TCAGGATGAT CTGGACGAAG AGCATCAGGG GCTCGCGCCA 
2461 GCCGAACTGT TCGCCAGGCT CAAGGCGCGC ATGCCCGACG GCGAGGATCT CGTCGTGACC 
2521 CATGGCGATG CCTGCTTGCC GAATATCATG GTGGAAAATG GCCGCTTTTC TGGATTCATC 
2581 GACTGTGGCC GGCTGGGTGT GGCGGACCGC TATCAGGACA TAGCGTTGGC TACCCGTGAT 
2641 ATTGCTGAAG AGCTTGGCGG CGAATGGGCT GACCGCTTCC TCGTGCTTTA CGGTATCGCC 
2701 GCTCCCGATT CGCAGCGCAT CGCCTTCTAT CGCCTTCTTG ACGAGTTCTT CTGAGGGGAT 
2761 CCGTCGACCT GCAGCCAAGC TTATCGATAA AATAAAAGAT TTTATTTAGT CTCCAGAAAA 
2821 AGGGGGGAAT GAAAGACCCC ACCTGTAGGT TTGGCAAGCT AGCTTAAGTA ACGCCATTTT 
2881 GCAAGGCATG GAAAATACAT AACTGAGAAT AGAGAAGTTC AGATCAAGGT TAGGAACAGA 
2941 GAGACAGCAG AATATGGGCC AAACAGGATA TCTGTGGTAA GCAGTTCCTG CCCCGGCTCA 
3001 GGGCCAAGAA CAGATGGTCC CCAGATGCGG TCCCGCCCTC AGCAGTTTCT AGAGAACCAT 
3061 CAGATGTTTC CAGGGTGCCC CAAGGACCTG AAATGACCCT GTGCCTTATT TGAACTAACC 
3121 AATCAGTTCG CTTCTCGCTT CTGTTCGCGC GCTTCTGCTC CCCGAGCTCA ATAAAAGAGC 
3181 CCACAACCCC TCACTCGGCG CGCCAGTCCT CCGATAGACT GCGTCGCCCG GGTACCCGTG 
3241 TATCCAATAA ACCCTCTTGC AGTTGCATCC GACTTGTGGT CTCGCTGTTC CTTGGGAGGG 
3301 TCTCCTCTGA GTGATTGACT ACCCGTCAGC GGGGGTCTTT CATGGGTAAC AGTTTCTTGA 
3361 AGTTGGAGAA CAACATTCTG AGGGTAGGAG TCGAATATTA AGTAATCCTG ACTCAATTAG 
3421 CCACTGTTTT GAATCCACAT ACTCCAATAC TCCTGAAATA GTTCATTATG GACAGCGCAG 
3481 AAAGAGCTGG GGAGAATTGT GAAATTGTTA TCCGCTCACA ATTCCACACA ACATACGAGC 
3541 CGGAAGCATA AAGTGTAAAG CCTGGGGTGC CTAATGAGTG AGCTAACTCA CATTAATTGC 
3601 GTTGCGCTCA CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT 
3661 CGGCCAACGC GCGGGGAGAG GCGGTTTGCG TATTGGGCGC TCTTCCGCTT CCTCGCTCAC 
3721 TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT 
3781 AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG CAAAAGGCCA 
3841 GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC 
3901 CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT 
3961 ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT 
4021 GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG 
4081 CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA 
4141 CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA 
4201 CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC 
4261 GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG 
4321 AAGAACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG 
4381 TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA 
4441 GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC 
4501 TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG 
4561 GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA 
4621 TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT 
4681 CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA CTACGATACG 
4741 GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCG CGAGACCCAC GCTCACCGGC 
4801 TCCAGATTTA TCAGCAATAA ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC 
4861 AACTTTATCC GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC 
4921 GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTACA GGCATCGTGG TGTCACGCTC 
4981 GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG TTACATGATC 
5041 CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT CCGATCGTTG TCAGAAGTAA 
5101 GTTGGCCGCA GTGTTATCAC TCATGGTTAT GGCAGCACTG CATAATTCTC TTACTGTCAT 
5161 GCCATCCGTA AGATGCTTTT CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA 
5221 GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAATA CGGGATAATA CCGCGCCACA 
5281 TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA AACTCTCAAG 
5341 GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA ACTGATCTTC 
5401 AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC AAAATGCCGC 
5461 AAAAAAGGGA ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA 
5521 TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG AATGTATTTA 
5581 GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC CTGACGTCTA 
5641 AGAAACCATT ATTATCATGA CATTAACCTA TAAAAATAGG CGTATCACGA GGCCCTTTCG 
5701 TCTCGCGCGT TTCGGTGATG ACGGTGAAAA CCTCTGACAC ATGCAGCTCC CGGAGACGGT 
5761 CACAGCTTGT CTGTAAGCGG ATGCCGGGAG CAGACAAGCC CGTCAGGGCG CGTCAGCGGG 
5821 TGTTGGCGGG TGTCGGGGCT GGCTTAACTA TGCGGCATCA GAGCAGATTG TACTGAGAGT 
5881 GCACCATATG CGGTGTGAAA TACCGCACAG ATGCGTAAGG AGAAAATACC GCATCAGGCG 
5941 CCATTCGCCA TTCAGGCTGC GCAACTGTTG GGAAGGGCGA TCGGTGCGGG CCTCTTCGCT 
6001 ATTACGCCAG CTGGCGAAAG GGGGATGTGC TGCAAGGCGA TTAAGTTGGG TAACGCCAGG 
6061 GTTTTCCCAG TCACGACGTT GTAAAACGAC GGCGCAAGGA ATGGTGCATG CAAGGAGATG 
6121 GCGCCCAACA GTCCCCCGGC CACGGGGCCT GCCACCATAC CCACGCCGAA ACAAGCGCTC 
6181 ATGAGCCCGA AGTGGCGAGC CCGATCTTCC CCATCGGTGA TGTCGGCGAT ATAGGCGCCA 
6241 GCAACCGCAC CTGTGGCGCC GGTGATGCCG GCCACGATGC GTCCGGCGTA GAGGCGATTA 
6301 GTCCAATTTG TTAAAGACAG GATATCAGTG GTCCAGGCTC TAGTTTTGAC TCAACAATAT 
6361 CACCAGCTGA AGCCTATAGA GTACGAGCCA TAGATAAAAT AAAAGATTTT ATTTAGTCTC 
6421 CAGAAAAAGG GGGGAA