pLVX-IRES-mCherry is an HIV-1-based, lentiviral expression vector that allows the simultaneous expression of your protein of interest and mCherry in virtually any mammalian cell type, including primary cells. mCherry is a mutant fluorescent protein derived from the tetrameric Discosoma sp. red fluorescent protein, DsRed (1). The vector expresses the two proteins from a bicistronic mRNA transcript, allowing mCherry to be used as an indicator of transduction efficiency and a marker for selection by flow cytometry.

Expression of the bicistronic transcript is driven by the constitutively active human cytomegalovirus immediate early promoter (PCMV IE) located just upstream of the MCS. An encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), positioned between the MCS and mCherry, facilitates cap-independent translation of mCherry from an internal start site at the IRES/mCherry junction (1).

pLVX-IRES-mCherry contains all of the viral processing elements necessary for the production of replication-incompetent lentivirus, as well as elements to improve viral titer, transgene expression, and overall vector function. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) promotes RNA processing events and enhances nuclear export of viral RNA (2), leading to increased viral titers from packaging cells. In addition, the vector includes a Rev-response element (RRE), which further increases viral titers by enhancing the transport of unspliced viral RNA out of the nucleus (3). Finally, pLVX-IRES-mCherry also contains a central polypurine tract/central termination sequence element (cPPT/CTS). During target cell infection, this element creates a central DNA flap that increases nuclear import of the viral genome, resulting in improved vector integration and more efficient transduction (4). The vector also contains a pUC origin of replication and an E. coli ampicillin resistance gene (Ampr) for propagation and selection in bacteria.

載體應(yīng)用

pLVX-IRES-mCherry is designed to constitutively coexpress your protein of interest and mCherry from PCMV IE when transduced into mammalian cells. Before it can be transduced into target cells, the vector must be packaged into viral particles in HEK293T cells, using our Lenti-X? HT Packaging System (Cat. Nos. 632160 and 632161). This packaging system allows the safe production of high titer, infectious, replication-incompetent, VSV-G pseudotyped lentiviral particles that can infect a wide range of cell types, including nondividing and primary cells (5).

The presence of mCherry allows transductants to be visualized by fluorescence microscopy and sorted by flow cytometry with standard FITC filter sets (mCherry has an excitation maximum of 587 nm and an emission maximum of 610 nm).

0001 TGGAAGGGCT AATTCACTCC CAAAGAAGAC AAGATATCCT TGATCTGTGG ATCTACCACA 
0061 CACAAGGCTA CTTCCCTGAT TAGCAGAACT ACACACCAGG GCCAGGGGTC AGATATCCAC 
0121 TGACCTTTGG ATGGTGCTAC AAGCTAGTAC CAGTTGAGCC AGATAAGGTA GAAGAGGCCA 
0181 ATAAAGGAGA GAACACCAGC TTGTTACACC CTGTGAGCCT GCATGGGATG GATGACCCGG 
0241 AGAGAGAAGT GTTAGAGTGG AGGTTTGACA GCCGCCTAGC ATTTCATCAC GTGGCCCGAG 
0301 AGCTGCATCC GGAGTACTTC AAGAACTGCT GATATCGAGC TTGCTACAAG GGACTTTCCG 
0361 CTGGGGACTT TCCAGGGAGG CGTGGCCTGG GCGGGACTGG GGAGTGGCGA GCCCTCAGAT 
0421 CCTGCATATA AGCAGCTGCT TTTTGCCTGT ACTGGGTCTC TCTGGTTAGA CCAGATCTGA 
0481 GCCTGGGAGC TCTCTGGCTA ACTAGGGAAC CCACTGCTTA AGCCTCAATA AAGCTTGCCT 
0541 TGAGTGCTTC AAGTAGTGTG TGCCCGTCTG TTGTGTGACT CTGGTAACTA GAGATCCCTC 
0601 AGACCCTTTT AGTCAGTGTG GAAAATCTCT AGCAGTGGCG CCCGAACAGG GACTTGAAAG 
0661 CGAAAGGGAA ACCAGAGGAG CTCTCTCGAC GCAGGACTCG GCTTGCTGAA GCGCGCACGG 
0721 CAAGAGGCGA GGGGCGGCGA CTGGTGAGTA CGCCAAAAAT TTTGACTAGC GGAGGCTAGA 
0781 AGGAGAGAGA TGGGTGCGAG AGCGTCAGTA TTAAGCGGGG GAGAATTAGA TCGCGATGGG 
0841 AAAAAATTCG GTTAAGGCCA GGGGGAAAGA AAAAATATAA ATTAAAACAT ATAGTATGGG 
0901 CAAGCAGGGA GCTAGAACGA TTCGCAGTTA ATCCTGGCCT GTTAGAAACA TCAGAAGGCT 
0961 GTAGACAAAT ACTGGGACAG CTACAACCAT CCCTTCAGAC AGGATCAGAA GAACTTAGAT 
1021 CATTATATAA TACAGTAGCA ACCCTCTATT GTGTGCATCA AAGGATAGAG ATAAAAGACA 
1081 CCAAGGAAGC TTTAGACAAG ATAGAGGAAG AGCAAAACAA AAGTAAGACC ACCGCACAGC 
1141 AAGCGGCCGG CCGCTGATCT TCAGACCTGG AGGAGGAGAT ATGAGGGACA ATTGGAGAAG 
1201 TGAATTATAT AAATATAAAG TAGTAAAAAT TGAACCATTA GGAGTAGCAC CCACCAAGGC 
1261 AAAGAGAAGA GTGGTGCAGA GAGAAAAAAG AGCAGTGGGA ATAGGAGCTT TGTTCCTTGG 
1321 GTTCTTGGGA GCAGCAGGAA GCACTATGGG CGCAGCGTCA ATGACGCTGA CGGTACAGGC 
1381 CAGACAATTA TTGTCTGGTA TAGTGCAGCA GCAGAACAAT TTGCTGAGGG CTATTGAGGC 
1441 GCAACAGCAT CTGTTGCAAC TCACAGTCTG GGGCATCAAG CAGCTCCAGG CAAGAATCCT 
1501 GGCTGTGGAA AGATACCTAA AGGATCAACA GCTCCTGGGG ATTTGGGGTT GCTCTGGAAA 
1561 ACTCATTTGC ACCACTGCTG TGCCTTGGAA TGCTAGTTGG AGTAATAAAT CTCTGGAACA 
1621 GATTTGGAAT CACACGACCT GGATGGAGTG GGACAGAGAA ATTAACAATT ACACAAGCTT 
1681 AATACACTCC TTAATTGAAG AATCGCAAAA CCAGCAAGAA AAGAATGAAC AAGAATTATT 
1741 GGAATTAGAT AAATGGGCAA GTTTGTGGAA TTGGTTTAAC ATAACAAATT GGCTGTGGTA 
1801 TATAAAATTA TTCATAATGA TAGTAGGAGG CTTGGTAGGT TTAAGAATAG TTTTTGCTGT 
1861 ACTTTCTATA GTGAATAGAG TTAGGCAGGG ATATTCACCA TTATCGTTTC AGACCCACCT 
1921 CCCAACCCCG AGGGGACCCG ACAGGCCCGA AGGAATAGAA GAAGAAGGTG GAGAGAGAGA 
1981 CAGAGACAGA TCCATTCGAT TAGTGAACGG ATCTCGACGG TATCGCCTTT AAAAGAAAAG 
2041 GGGGGATTGG GGGGTACAGT GCAGGGGAAA GAATAGTAGA CATAATAGCA ACAGACATAC 
2101 AAACTAAAGA ATTACAAAAA CAAATTACAA AAATTCAAAA TTTTCGGGTT TATTACAGGG 
2161 ACAGCAGAGA TCCAGTTTAT CGATAAGCTT GGGAGTTCCG CGTTACATAA CTTACGGTAA 
2221 ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG 
2281 TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT 
2341 AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG 
2401 TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC 
2461 CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC 
2521 AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA 
2581 TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA 
2641 ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATATAA 
2701 GCAGAGCTCG TTTAGTGAAC CGTCAGATCG CCTGGAGACG CCATCCACGC TGTTTTGACC 
2761 TCCATAGAAG ACACCGACTC TACTAGAGGA TCTATTTCCG GTGAATTCCT CGAGACTAGT 
2821 TCTAGAGCGG CCGCGGATCC CGCCCCTCTC CCTCCCCCCC CCCTAACGTT ACTGGCCGAA 
2881 GCCGCTTGGA ATAAGGCCGG TGTGCGTTTG TCTATATGTT ATTTTCCACC ATATTGCCGT 
2941 CTTTTGGCAA TGTGAGGGCC CGGAAACCTG GCCCTGTCTT CTTGACGAGC ATTCCTAGGG 
3001 GTCTTTCCCC TCTCGCCAAA GGAATGCAAG GTCTGTTGAA TGTCGTGAAG GAAGCAGTTC 
3061 CTCTGGAAGC TTCTTGAAGA CAAACAACGT CTGTAGCGAC CCTTTGCAGG CAGCGGAACC 
3121 CCCCACCTGG CGACAGGTGC CTCTGCGGCC AAAAGCCACG TGTATAAGAT ACACCTGCAA 
3181 AGGCGGCACA ACCCCAGTGC CACGTTGTGA GTTGGATAGT TGTGGAAAGA GTCAAATGGC 
3241 TCACCTCAAG CGTATTCAAC AAGGGGCTGA AGGATGCCCA GAAGGTACCC CATTGTATGG 
3301 GATCTGATCT GGGGCCTCGG TGCACATGCT TTACATGTGT TTAGTCGAGG TTAAAAAACG 
3361 TCTAGGCCCC CCGAACCACG GGGACGTGGT TTTCCTTTGA AAAACACGAT GATAATATGG 
3421 TGAGCAAGGG CGAGGAGGAT AACATGGCCA TCATCAAGGA GTTCATGCGC TTCAAGGTGC 
3481 ACATGGAGGG CTCCGTGAAC GGCCACGAGT TCGAGATCGA GGGCGAGGGC GAGGGCCGCC 
3541 CCTACGAGGG CACCCAGACC GCCAAGCTGA AGGTGACCAA GGGTGGCCCC CTGCCCTTCG 
3601 CCTGGGACAT CCTGTCCCCT CAGTTCATGT ACGGCTCCAA GGCCTACGTG AAGCACCCCG 
3661 CCGACATCCC CGACTACTTG AAGCTGTCCT TCCCCGAGGG CTTCAAGTGG GAGCGCGTGA 
3721 TGAACTTCGA GGACGGCGGC GTGGTGACCG TGACCCAGGA CTCCTCCCTG CAGGACGGCG 
3781 AGTTCATCTA CAAGGTGAAG CTGCGCGGCA CCAACTTCCC CTCCGACGGC CCCGTAATGC 
3841 AGAAGAAGAC CATGGGCTGG GAGGCCTCCT CCGAGCGGAT GTACCCCGAG GACGGCGCCC 
3901 TGAAGGGCGA GATCAAGCAG AGGCTGAAGC TGAAGGACGG CGGCCACTAC GACGCTGAGG 
3961 TCAAGACCAC CTACAAGGCC AAGAAGCCCG TGCAGCTGCC CGGCGCCTAC AACGTCAACA 
4021 TCAAGTTGGA CATCACCTCC CACAACGAGG ACTACACCAT CGTGGAACAG TACGAACGCG 
4081 CCGAGGGCCG CCACTCCACC GGCGGCATGG ACGAGCTGTA CAAGTGAACG CGTCTGGAAC 
4141 AATCAACCTC TGGATTACAA AATTTGTGAA AGATTGACTG GTATTCTTAA CTATGTTGCT 
4201 CCTTTTACGC TATGTGGATA CGCTGCTTTA ATGCCTTTGT ATCATGCTAT TGCTTCCCGT 
4261 ATGGCTTTCA TTTTCTCCTC CTTGTATAAA TCCTGGTTGC TGTCTCTTTA TGAGGAGTTG 
4321 TGGCCCGTTG TCAGGCAACG TGGCGTGGTG TGCACTGTGT TTGCTGACGC AACCCCCACT 
4381 GGTTGGGGCA TTGCCACCAC CTGTCAGCTC CTTTCCGGGA CTTTCGCTTT CCCCCTCCCT 
4441 ATTGCCACGG CGGAACTCAT CGCCGCCTGC CTTGCCCGCT GCTGGACAGG GGCTCGGCTG 
4501 TTGGGCACTG ACAATTCCGT GGTGTTGTCG GGGAAGCTGA CGTCCTTTCC ATGGCTGCTC 
4561 GCCTGTGTTG CCACCTGGAT TCTGCGCGGG ACGTCCTTCT GCTACGTCCC TTCGGCCCTC 
4621 AATCCAGCGG ACCTTCCTTC CCGCGGCCTG CTGCCGGCTC TGCGGCCTCT TCCGCGTCTT 
4681 CGCCTTCGCC CTCAGACGAG TCGGATCTCC CTTTGGGCCG CCTCCCCGCC TGGAATTAAT 
4741 TCTGCAGTCG AGACCTAGAA AAACATGGAG CAATCACAAG TAGCAATACA GCAGCTACCA 
4801 ATGCTGATTG TGCCTGGCTA GAAGCACAAG AGGAGGAGGA GGTGGGTTTT CCAGTCACAC 
4861 CTCAGGTACC TTTAAGACCA ATGACTTACA AGGCAGCTGT AGATCTTAGC CACTTTTTAA 
4921 AAGAAAAGAG GGGACTGGAA GGGCTAATTC ACTCCCAACG AAGACAAGAT ATCCTTGATC 
4981 TGTGGATCTA CCACACACAA GGCTACTTCC CTGATTAGCA GAACTACACA CCAGGGCCAG 
5041 GGGTCAGATA TCCACTGACC TTTGGATGGT GCTACAAGCT AGTACCAGTT GAGCCAGATA 
5101 AGGTAGAAGA GGCCAATAAA GGAGAGAACA CCAGCTTGTT ACACCCTGTG AGCCTGCATG 
5161 GGATGGATGA CCCGGAGAGA GAAGTGTTAG AGTGGAGGTT TGACAGCCGC CTAGCATTTC 
5221 ATCACGTGGC CCGAGAGCTG CATCCGGAGT ACTTCAAGAA CTGCTGATAT CGAGCTTGCT 
5281 ACAAGGGACT TTCCGCTGGG GACTTTCCAG GGAGGCGTGG CCTGGGCGGG ACTGGGGAGT 
5341 GGCGAGCCCT CAGATCCTGC ATATAAGCAG CTGCTTTTTG CCTGTACTGG GTCTCTCTGG 
5401 TTAGACCAGA TCTGAGCCTG GGAGCTCTCT GGCTAACTAG GGAACCCACT GCTTAAGCCT 
5461 CAATAAAGCT TGCCTTGAGT GCTTCAAGTA GTGTGTGCCC GTCTGTTGTG TGACTCTGGT 
5521 AACTAGAGAT CCCTCAGACC CTTTTAGTCA GTGTGGAAAA TCTCTAGCAG TAGTAGTTCA 
5581 TGTCATCTTA TTATTCAGTA TTTATAACTT GCAAAGAAAT GAATATCAGA GAGTGAGAGG 
5641 CCTTGACATT GCTAGCGTTT ACCGTCGACC TCTAGCTAGA GCTTGGCGTA ATCATGGTCA 
5701 TAGCTGTTTC CTGTGTGAAA TTGTTATCCG CTCACAATTC CACACAACAT ACGAGCCGGA 
5761 AGCATAAAGT GTAAAGCCTG GGGTGCCTAA TGAGTGAGCT AACTCACATT AATTGCGTTG 
5821 CGCTCACTGC CCGCTTTCCA GTCGGGAAAC CTGTCGTGCC AGCTGCATTA ATGAATCGGC 
5881 CAACGCGCGG GGAGAGGCGG TTTGCGTATT GGGCGCTCTT CCGCTTCCTC GCTCACTGAC 
5941 TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA GCGGTATCAG CTCACTCAAA GGCGGTAATA 
6001 CGGTTATCCA CAGAATCAGG GGATAACGCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA 
6061 AAGGCCAGGA ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT 
6121 GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC GAAACCCGAC AGGACTATAA 
6181 AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG 
6241 CTTACCGGAT ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC TCATAGCTCA 
6301 CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA 
6361 CCCCCCGTTC AGCCCGACCG CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG 
6421 GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA ACAGGATTAG CAGAGCGAGG 
6481 TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA ACTACGGCTA CACTAGAAGA 
6541 ACAGTATTTG GTATCTGCGC TCTGCTGAAG CCAGTTACCT TCGGAAAAAG AGTTGGTAGC 
6601 TCTTGATCCG GCAAACAAAC CACCGCTGGT AGCGGTGGTT TTTTTGTTTG CAAGCAGCAG 
6661 ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA TCTTTTCTAC GGGGTCTGAC 
6721 GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC 
6781 TTCACCTAGA TCCTTTTAAA TTAAAAATGA AGTTTTAAAT CAATCTAAAG TATATATGAG 
6841 TAAACTTGGT CTGACAGTTA CCAATGCTTA ATCAGTGAGG CACCTATCTC AGCGATCTGT 
6901 CTATTTCGTT CATCCATAGT TGCCTGACTC CCCGTCGTGT AGATAACTAC GATACGGGAG 
6961 GGCTTACCAT CTGGCCCCAG TGCTGCAATG ATACCGCGAG ACCCACGCTC ACCGGCTCCA 
7021 GATTTATCAG CAATAAACCA GCCAGCCGGA AGGGCCGAGC GCAGAAGTGG TCCTGCAACT 
7081 TTATCCGCCT CCATCCAGTC TATTAATTGT TGCCGGGAAG CTAGAGTAAG TAGTTCGCCA 
7141 GTTAATAGTT TGCGCAACGT TGTTGCCATT GCTACAGGCA TCGTGGTGTC ACGCTCGTCG 
7201 TTTGGTATGG CTTCATTCAG CTCCGGTTCC CAACGATCAA GGCGAGTTAC ATGATCCCCC 
7261 ATGTTGTGCA AAAAAGCGGT TAGCTCCTTC GGTCCTCCGA TCGTTGTCAG AAGTAAGTTG 
7321 GCCGCAGTGT TATCACTCAT GGTTATGGCA GCACTGCATA ATTCTCTTAC TGTCATGCCA 
7381 TCCGTAAGAT GCTTTTCTGT GACTGGTGAG TACTCAACCA AGTCATTCTG AGAATAGTGT 
7441 ATGCGGCGAC CGAGTTGCTC TTGCCCGGCG TCAATACGGG ATAATACCGC GCCACATAGC 
7501 AGAACTTTAA AAGTGCTCAT CATTGGAAAA CGTTCTTCGG GGCGAAAACT CTCAAGGATC 
7561 TTACCGCTGT TGAGATCCAG TTCGATGTAA CCCACTCGTG CACCCAACTG ATCTTCAGCA 
7621 TCTTTTACTT TCACCAGCGT TTCTGGGTGA GCAAAAACAG GAAGGCAAAA TGCCGCAAAA 
7681 AAGGGAATAA GGGCGACACG GAAATGTTGA ATACTCATAC TCTTCCTTTT TCAATATTAT 
7741 TGAAGCATTT ATCAGGGTTA TTGTCTCATG AGCGGATACA TATTTGAATG TATTTAGAAA 
7801 AATAAACAAA TAGGGGTTCC GCGCACATTT CCCCGAAAAG TGCCACCTGA CGTCGACGGA 
7861 TCGGGAGATC AACTTGTTTA TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC 
7921 AAATTTCACA AATAAAGCAT TTTTTTCACT GCATTCTAGT TGTGGTTTGT CCAAACTCAT 
7981 CAATGTATCT TATCATGTCT GGATCAACTG GATAACTCAA GCTAACCAAA ATCATCCCAA 
8041 ACTTCCCACC CCATACCCTA TTACCACTGC CAATTACCTG TGGTTTCATT TACTCTAAAC 
8101 CTGTGATTCC TCTGAATTAT TTTCATTTTA AAGAAATTGT ATTTGTTAAA TATGTACTAC 
8161 AAACTTAGTA GT