pMSCVhyg

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, pMSCVhyg transiently expresses, or integrates and stably expresses, a transcript containing the extended viral packaging signal !+, the hygromycin 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), when cloned into the multiple cloning site downstream of the 5' LTR. The murine phosphoglycerate kinase (PKG) promoter (PPKG) controls expression of the hygromycin resistance gene (Hygr) for antibiotic selection in eukaryotic cells. pMSCVhyg also contains the pUC origin of replication and E. coli Ampr gene for propagation and antibiotic selection in bacteria.

载体应用

pMSCVhyg 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. pMSCVhyg 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 pMSCVhyg 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 TGCATCCCGC CACCATGAAA AAGCCTGAAC TCACCGCGAC 
1981 GTCTGTCGAG AAGTTTCTGA TCGAAAAGTT CGACAGCGTC TCCGACCTGA TGCAGCTCTC 
2041 GGAGGGCGAA GAATCTCGTG CTTTCAGCTT CGATGTAGGA GGGCGTGGAT ATGTCCTGCG 
2101 GGTAAATAGC TGCGCCGATG GTTTCTACAA AGATCGTTAT GTTTATCGGC ACTTTGCATC 
2161 GGCCGCGCTC CCGATTCCGG AAGTGCTTGA CATTGGGGAA TTCAGCGAGA GCCTGACCTA 
2221 TTGCATCTCC CGCCGTGCAC AGGGTGTCAC GTTGCAAGAC CTGCCTGAAA CCGAACTGCC 
2281 CGCTGTTCTG CAGCCGGTCG CGGAGGCCAT GGATGCGATC GCTGCGGCCG ATCTTAGCCA 
2341 GACGAGCGGG TTCGGCCCAT TCGGACCGCA AGGAATCGGT CAATACACTA CATGGCGTGA 
2401 TTTCATATGC GCGATTGCTG ATCCCCATGT GTATCACTGG CAAACTGTGA TGGACGACAC 
2461 CGTCAGTGCG TCCGTCGCGC AGGCTCTCGA TGAGCTGATG CTTTGGGCCG AGGACTGCCC 
2521 CGAAGTCCGG CACCTCGTGC ACGCGGATTT CGGCTCCAAC AATGTCCTGA CGGACAATGG 
2581 CCGCATAACA GCGGTCATTG ACTGGAGCGA GGCGATGTTC GGGGATTCCC AATACGAGGT 
2641 CGCCAACATC TTCTTCTGGA GGCCGTGGTT GGCTTGTATG GAGCAGCAGA CGCGCTACTT 
2701 CGAGCGGAGG CATCCGGAGC TTGCAGGATC GCCGCGGCTC CGGGCGTATA TGCTCCGCAT 
2761 TGGTCTTGAC CAACTCTATC AGAGCTTGGT TGACGGCAAT TTCGATGATG CAGCTTGGGC 
2821 GCAGGGTCGA TGCGACGCAA TCGTCCGATC CGGAGCCGGG ACTGTCGGGC GTACACAAAT 
2881 CGCCCGCAGA AGCGCGGCCG TCTGGACCGA TGGCTGTGTA GAAGTACTCG CCGATAGTGG 
2941 AAACCGACGC CCCAGCACTC GTCCGAGGGC AAAGGAATAG AGTAGATGCC GACCGAACAA 
3001 GAGCTGATTT CGAGAACGCC TCAGCCAGCA ACTCGCGCGA GCCTAGCAAG GCAAATGCGA 
3061 GAGAACGGCC TTACGCTTGG TGGCACAGTT CTCGTCCACA GTTCGCTAAG CTCGCTCGGC 
3121 TGGGTCGCGG GAGGGCCGGT CGCAGTGATT CAGGCCCTTC TGGATTGTGT TGGTCCCCAG 
3181 GGCACGATTG TCATGCCCAC GCACTCGGGT GATCTGACTG ATCCCGCAGA TTGGAGATCG 
3241 CCGCCCGTGC CTGCCGATTG GGTGCAGATC CGTCGACCTG CAGCCAAGCT TATCGATAAA 
3301 ATAAAAGATT TTATTTAGTC TCCAGAAAAA GGGGGGAATG AAAGACCCCA CCTGTAGGTT 
3361 TGGCAAGCTA GCTTAAGTAA CGCCATTTTG CAAGGCATGG AAAATACATA ACTGAGAATA 
3421 GAGAAGTTCA GATCAAGGTT AGGAACAGAG AGACAGCAGA ATATGGGCCA AACAGGATAT 
3481 CTGTGGTAAG CAGTTCCTGC CCCGGCTCAG GGCCAAGAAC AGATGGTCCC CAGATGCGGT 
3541 CCCGCCCTCA GCAGTTTCTA GAGAACCATC AGATGTTTCC AGGGTGCCCC AAGGACCTGA 
3601 AATGACCCTG TGCCTTATTT GAACTAACCA ATCAGTTCGC TTCTCGCTTC TGTTCGCGCG 
3661 CTTCTGCTCC CCGAGCTCAA TAAAAGAGCC CACAACCCCT CACTCGGCGC GCCAGTCCTC 
3721 CGATAGACTG CGTCGCCCGG GTACCCGTGT ATCCAATAAA CCCTCTTGCA GTTGCATCCG 
3781 ACTTGTGGTC TCGCTGTTCC TTGGGAGGGT CTCCTCTGAG TGATTGACTA CCCGTCAGCG 
3841 GGGGTCTTTC ATGGGTAACA GTTTCTTGAA GTTGGAGAAC AACATTCTGA GGGTAGGAGT 
3901 CGAATATTAA GTAATCCTGA CTCAATTAGC CACTGTTTTG AATCCACATA CTCCAATACT 
3961 CCTGAAATAG TTCATTATGG ACAGCGCAGA AAGAGCTGGG GAGAATTGTG AAATTGTTAT 
4021 CCGCTCACAA TTCCACACAA CATACGAGCC GGAAGCATAA AGTGTAAAGC CTGGGGTGCC 
4081 TAATGAGTGA GCTAACTCAC ATTAATTGCG TTGCGCTCAC TGCCCGCTTT CCAGTCGGGA 
4141 AACCTGTCGT GCCAGCTGCA TTAATGAATC GGCCAACGCG CGGGGAGAGG CGGTTTGCGT 
4201 ATTGGGCGCT CTTCCGCTTC CTCGCTCACT GACTCGCTGC GCTCGGTCGT TCGGCTGCGG 
4261 CGAGCGGTAT CAGCTCACTC AAAGGCGGTA ATACGGTTAT CCACAGAATC AGGGGATAAC 
4321 GCAGGAAAGA ACATGTGAGC AAAAGGCCAG CAAAAGGCCA GGAACCGTAA AAAGGCCGCG 
4381 TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CCTGACGAGC ATCACAAAAA TCGACGCTCA 
4441 AGTCAGAGGT GGCGAAACCC GACAGGACTA TAAAGATACC AGGCGTTTCC CCCTGGAAGC 
4501 TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC CGCCTTTCTC 
4561 CCTTCGGGAA GCGTGGCGCT TTCTCATAGC TCACGCTGTA GGTATCTCAG TTCGGTGTAG 
4621 GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG TTCAGCCCGA CCGCTGCGCC 
4681 TTATCCGGTA ACTATCGTCT TGAGTCCAAC CCGGTAAGAC ACGACTTATC GCCACTGGCA 
4741 GCAGCCACTG GTAACAGGAT TAGCAGAGCG AGGTATGTAG GCGGTGCTAC AGAGTTCTTG 
4801 AAGTGGTGGC CTAACTACGG CTACACTAGA AGAACAGTAT TTGGTATCTG CGCTCTGCTG 
4861 AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT CCGGCAAACA AACCACCGCT 
4921 GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG CAGATTACGC GCAGAAAAAA AGGATCTCAA 
4981 GAAGATCCTT TGATCTTTTC TACGGGGTCT GACGCTCAGT GGAACGAAAA CTCACGTTAA 
5041 GGGATTTTGG TCATGAGATT ATCAAAAAGG ATCTTCACCT AGATCCTTTT AAATTAAAAA 
5101 TGAAGTTTTA AATCAATCTA AAGTATATAT GAGTAAACTT GGTCTGACAG TTACCAATGC 
5161 TTAATCAGTG AGGCACCTAT CTCAGCGATC TGTCTATTTC GTTCATCCAT AGTTGCCTGA 
5221 CTCCCCGTCG TGTAGATAAC TACGATACGG GAGGGCTTAC CATCTGGCCC CAGTGCTGCA 
5281 ATGATACCGC GAGACCCACG CTCACCGGCT CCAGATTTAT CAGCAATAAA CCAGCCAGCC 
5341 GGAAGGGCCG AGCGCAGAAG TGGTCCTGCA ACTTTATCCG CCTCCATCCA GTCTATTAAT 
5401 TGTTGCCGGG AAGCTAGAGT AAGTAGTTCG CCAGTTAATA GTTTGCGCAA CGTTGTTGCC 
5461 ATTGCTACAG GCATCGTGGT GTCACGCTCG TCGTTTGGTA TGGCTTCATT CAGCTCCGGT 
5521 TCCCAACGAT CAAGGCGAGT TACATGATCC CCCATGTTGT GCAAAAAAGC GGTTAGCTCC 
5581 TTCGGTCCTC CGATCGTTGT CAGAAGTAAG TTGGCCGCAG TGTTATCACT CATGGTTATG 
5641 GCAGCACTGC ATAATTCTCT TACTGTCATG CCATCCGTAA GATGCTTTTC TGTGACTGGT 
5701 GAGTACTCAA CCAAGTCATT CTGAGAATAG TGTATGCGGC GACCGAGTTG CTCTTGCCCG 
5761 GCGTCAATAC GGGATAATAC CGCGCCACAT AGCAGAACTT TAAAAGTGCT CATCATTGGA 
5821 AAACGTTCTT CGGGGCGAAA ACTCTCAAGG ATCTTACCGC TGTTGAGATC CAGTTCGATG 
5881 TAACCCACTC GTGCACCCAA CTGATCTTCA GCATCTTTTA CTTTCACCAG CGTTTCTGGG 
5941 TGAGCAAAAA CAGGAAGGCA AAATGCCGCA AAAAAGGGAA TAAGGGCGAC ACGGAAATGT 
6001 TGAATACTCA TACTCTTCCT TTTTCAATAT TATTGAAGCA TTTATCAGGG TTATTGTCTC 
6061 ATGAGCGGAT ACATATTTGA ATGTATTTAG AAAAATAAAC AAATAGGGGT TCCGCGCACA 
6121 TTTCCCCGAA AAGTGCCACC TGACGTCTAA GAAACCATTA TTATCATGAC ATTAACCTAT 
6181 AAAAATAGGC GTATCACGAG GCCCTTTCGT CTCGCGCGTT TCGGTGATGA CGGTGAAAAC 
6241 CTCTGACACA TGCAGCTCCC GGAGACGGTC ACAGCTTGTC TGTAAGCGGA TGCCGGGAGC 
6301 AGACAAGCCC GTCAGGGCGC GTCAGCGGGT GTTGGCGGGT GTCGGGGCTG GCTTAACTAT 
6361 GCGGCATCAG AGCAGATTGT ACTGAGAGTG CACCATATGC GGTGTGAAAT ACCGCACAGA 
6421 TGCGTAAGGA GAAAATACCG CATCAGGCGC CATTCGCCAT TCAGGCTGCG CAACTGTTGG 
6481 GAAGGGCGAT CGGTGCGGGC CTCTTCGCTA TTACGCCAGC TGGCGAAAGG GGGATGTGCT 
6541 GCAAGGCGAT TAAGTTGGGT AACGCCAGGG TTTTCCCAGT CACGACGTTG TAAAACGACG 
6601 GCGCAAGGAA TGGTGCATGC AAGGAGATGG CGCCCAACAG TCCCCCGGCC ACGGGGCCTG 
6661 CCACCATACC CACGCCGAAA CAAGCGCTCA TGAGCCCGAA GTGGCGAGCC CGATCTTCCC 
6721 CATCGGTGAT GTCGGCGATA TAGGCGCCAG CAACCGCACC TGTGGCGCCG GTGATGCCGG 
6781 CCACGATGCG TCCGGCGTAG AGGCGATTAG TCCAATTTGT TAAAGACAGG ATATCAGTGG 
6841 TCCAGGCTCT AGTTTTGACT CAACAATATC ACCAGCTGAA GCCTATAGAG TACGAGCCAT 
6901 AGATAAAATA AAAGATTTTA TTTAGTCTCC AGAAAAAGGG GGGAA