pRetroX-IRES-DsRedExpress

pRetroX-IRES-DsRedExpress is a bicistronic, ? uorescent, retroviral vector that allows both a gene of interest and the DsRed-Express gene to be translated from a single bicistronic mRNA. pRetroX-IRES-DsRedExpress is designed for ef? cient delivery and selection (by ? ow cytometry or other methods) of stably transduced mammalian cells expressing the DsRed-Express ? uorescent protein and the protein of interest. This vector can be used to obtain stable cell lines without time-consuming drug and clonal selection. DsRed-Express is a rapidly maturing variant of Discosoma sp. red ? uorescent protein (DsRed) and is easily detected with standard Rhodamine/propidium iodide ? lter sets (1).

Bicistronic expression from this vector is facilitated by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). This IRES facilitates cap-independent translation from an internal start site at the IRES/DsRed-Express junction (2). DsRed-Express contains nine amino acid substitutions that enhance its solubility, reduce its green emission, and accelerate its maturation (3). This retroviral vector is derived from the pMIN series of vectors (4, 5). These optimized vectors have the ability to produce high viral titers, express genes at high levels, and, due to the absence of retroviral coding sequences, exhibit improved safety pro? les. The multiple cloning site (MCS) in pRetroX-IRES-DsRedExpress is between the 5’ MMLV LTR and the IRES sequence. Genes cloned into the MCS are expressed as a bicistronic message transcribed from the 5’ LTR.

pRetroX-IRES-DsRedExpress contains all of the necessary viral RNA processing elements; these include the 5’ and 3’ LTRs, the packaging signal (ψ), and the tRNA primer-binding site. For safety reasons, however, the vector lacks the structural genes (gag, pol, and env) necessary for retroviral particle formation and replication. pRetroX-IRES-DsRedExpress contains a ColE1 origin of replication, and an E. coli Ampr gene for propagation and selection in bacteria.

载体应用

pRetroX-IRES-DsRedExpress is designed to ef? ciently deliver and co-express your gene of interest and DsRed-Express in any mitotically-active mammalian cell. When cloning into pRetroX-IRES-DsRedExpress, your gene of interest should contain an initiation codon (ATG) and a stop codon. The vector can be infected or transfected into mammalian cells. If required, stable transformants can be selected by ? ow cytometry or limiting dilution.

In order to infect mammalian cells with pRetroX-IRES-DsRedExpress, the vector must be transfected into a packaging cell line, such as the RetroPack? PT67 Cell line (631510), AmphoPack?-293 (631505), EcoPack?2- 293 (631507), Pantropic Expression System (631512), or Retro-X? Universal Packaging System (631530). These cell lines package RNA from the vector into infectious, replication-incompetent, retroviral particles. Such retroviral particles can infect target cells and transmit the gene of interest, but cannot replicate within these cells due to the absence of viral structural genes. The separate introduction and integration of the structural genes into the packaging cell line minimizes the chance of producing replication-competent virus due to recombination events during cell proliferation.

0001 TGAAAGACCC CACCTGTAGG TTTGGCAAGC TAGCTTAAGT AACGCCATTT TGCAAGGCAT 
0061 GGAAAAATAC ATAACTGAGA ATAGAAAAGT TCAGATCAAG GTCAGGAACA GATGGAACAG 
0121 CTGAATATGG GCCAAACAGG ATATCTGTGG TAAGCAGTTC CTGCCCCGGC TCAGGGCCAA 
0181 GAACAGATGG AACAGCTGAA TATGGGCCAA ACAGGATATC TGTGGTAAGC AGTTCCTGCC 
0241 CCGGCTCAGG GCCAAGAACA GATGGTCCCC AGATGCGGTC CAGCCCTCAG CAGTTTCTAG 
0301 AGAACCATCA GATGTTTCCA GGGTGCCCCA AGGACCTGAA ATGACCCTGT GCCTTATTTG 
0361 AACTAACCAA TCAGTTCGCT TCTCGCTTCT GTTCGCGCGC TTCTGCTCCC CGGGCTCAAT 
0421 AAAAGAGCCC ACAACCCCTC ACTCGGCGCG CCAGTCCTCC GATTGACTGA GTCGCCCGGG 
0481 TACCCGTGTA TCCAATAAAC CCTCTTGCAG TTGCATCCGA CTTGTGGCCT CGCTGTTCCT 
0541 TGGGAGGGTC TCCTCTGAGT GATTGACTAC CCGTCAGCGG GGGTCTTTCA TTTGGGGGCT 
0601 CGTCCGGGAT CGGGAGACCC CTGCCCAGGG ACCACCGACC CACCACCGGG AGGTAAGCTG 
0661 GCCAGCAACT TATCTGTGTC TGTCCGATTG TCTAGTGTCT ATGACTGATT TTATGCGCCT 
0721 GCGTCGGTAC TAGTTAGCTA ACTAGCTCTG TATCTGGCGG ACCCGTGGTG GAACTGACGA 
0781 GTTCGGAACA CCCGGCCGCA ACCCTGGGAG ACGTCCCAGG GACTTCGGGG GCCGTTTTTG 
0841 TGGCCCGACC TGAGTCCAAA AATCCCGATC GTTTTGGACT CTTTGGTGCA CCCCCCTTAG 
0901 AGGAGGGATA TGTGGTTCTG GTAGGAGACG AGAACCTAAA ACAGTTCCCG CCTCCGTCTG 
0961 AATTTTTGCT TTCGGTTTGG GACCGAAGCC GCGCCGCGCG TCTTGTCTGC TGCAGCATCG 
1021 TTCTGTGTTG TCTCTGTCTG ACTGTGTTTC TGTATTTGTC TGAAAATACG CGTGTCGAGC 
1081 TTTTGGAGTA CGTCGTCTTT AGGTTGGGGG GAGGGGTTTT ATGCGATGGA GTTTCCCCAC 
1141 ACTGAGTGGG TGGAGACTGA AGTTAGGCCA GCTTGGCACT TGATGTAATT CTCCTTGGAA 
1201 TTTGCCCTTT TTGAGTTTGG ATCTTGGTTC ATTCTCAAGC CTCAGACAGT GGTTCAAAGT 
1261 TTTTTTCTTC CATTTCAGCC GTCGTGAAAA CTACCCCTAA AAGCCAAAAA AATCGAATTC 
1321 CCAAACTTAA GCTTGGTACC GAGCTCGGAT CTCACGTGGG CCCGCGGCCG CAGATCTATC 
1381 GATGGATCCG TGCCCCTCTC CCTCCCCCCC CCCTAACGTT ACTGGCCGAA GCCGCTTGGA 
1441 ATAAGGCCGG TGTGCGTTTG TCTATATGTT ATTTTCCACC ATATTGCCGT CTTTTGGCAA 
1501 TGTGAGGGCC CGGAAACCTG GCCCTGTCTT CTTGACGAGC ATTCCTAGGG GTCTTTCCCC 
1561 TCTCGCCAAA GGAATGCAAG GTCTGTTGAA TGTCGTGAAG GAAGCAGTTC CTCTGGAAGC 
1621 TTCTTGAAGA CAAACAACGT CTGTAGCGAC CCTTTGCAGG CAGCGGAACC CCCCACCTGG 
1681 CGACAGGTGC CTCTGCGGCC AAAAGCCACG TGTATAAGAT ACACCTGCAA AGGCGGCACA 
1741 ACCCCAGTGC CACGTTGTGA GTTGGATAGT TGTGGAAAGA GTCAAATGGC TCACCTCAAG 
1801 CGTATTCAAC AAGGGGCTGA AGGATGCCCA GAAGGTACCC CATTGTATGG GATCTGATCT 
1861 GGGGCCTCGG TGCACATGCT TTACATGTGT TTAGTCGAGG TTAAAAAACG TCTAGGCCCC 
1921 CCGAACCACG GGGACGTGGT TTTCCTTTGA AAAACACGAT GATAATATGG CCTCCTCCGA 
1981 GGACGTCATC AAGGAGTTCA TGCGCTTCAA GGTGCGCATG GAGGGCTCCG TGAACGGCCA 
2041 CGAGTTCGAG ATCGAGGGCG AGGGCGAGGG CCGCCCCTAC GAGGGCACCC AGACCGCCAA 
2101 GCTGAAGGTG ACCAAGGGCG GCCCCCTGCC CTTCGCCTGG GACATCCTGT CCCCCCAGTT 
2161 CCAGTACGGC TCCAAGGTGT ACGTGAAGCA CCCCGCCGAC ATCCCCGACT ACAAGAAGCT 
2221 GTCCTTCCCC GAGGGCTTCA AGTGGGAGCG CGTGATGAAC TTCGAGGACG GCGGCGTGGT 
2281 GACCGTGACC CAGGACTCCT CCCTGCAGGA CGGCTCCTTC ATCTACAAGG TGAAGTTCAT 
2341 CGGCGTGAAC TTCCCCTCCG ACGGCCCCGT AATGCAGAAG AAGACTATGG GCTGGGAGGC 
2401 CTCCACCGAG CGCCTGTACC CCCGCGACGG CGTGCTGAAG GGCGAGATCC ACAAGGCCCT 
2461 GAAGCTGAAG GACGGCGGCC ACTACCTGGT GGAGTTCAAG TCTATCTATA TGGCCAAGAA 
2521 GCCCGTGCAG CTGCCCGGCT ACTACTACGT GGACTCCAAG CTGGACATCA CCTCCCACAA 
2581 CGAGGACTAC ACCATCGTGG AGCAGTACGA GCGCGCCGAG GGCCGCCACC ACCTGTTCCT 
2641 GTAGAATGCT CGAGGATAAA ATAAAAGATT TTATTTAGTC TCCAGAAAAA GGGGGGAATG 
2701 AAAGACCCCA CCTGTAGGTT TGGCAAGCTA GCTTAAGTAA CGCCATTTTG CAAGGCATGG 
2761 AAAAATACAT AACTGAGAAT AGAGAAGTTC AGATCAAGGT CAGGAACAGA TGGAACAGCT 
2821 GAATATGGGC CAAACAGGAT ATCTGTGGTA AGCAGTTCCT GCCCCGGCTC AGGGCCAAGA 
2881 ACAGATGGAA CAGCTGAATA TGGGCCAAAC AGGATATCTG TGGTAAGCAG TTCCTGCCCC 
2941 GGCTCAGGGC CAAGAACAGA TGGTCCCCAG ATGCGGTCCA GCCCTCAGCA GTTTCTAGAG 
3001 AACCATCAGA TGTTTCCAGG GTGCCCCAAG GACCTGAAAT GACCCTGTGC CTTATTTGAA 
3061 CTAACCAATC AGTTCGCTTC TCGCTTCTGT TCGCGCGCTT CTGCTCCCCG AGCTCAATAA 
3121 AAGAGCCCAC AACCCCTCAC TCGGGGCGCC AGTCCTCCGA TTGACTGAGT CGCCCGGGTA 
3181 CCCGTGTATC CAATAAACCC TCTTGCAGTT GCATCCGACT TGTGGTCTCG CTGTTCCTTG 
3241 GGAGGGTCTC CTCTGAGTGA TTGACTACCC GTCAGCGGGG GTCTTTCATT GAATTCGTAA 
3301 TCATGGTCAT AGCTGTTTCC TGTGTGAAAT TGTTATCCGC TCACAATTCC ACACAACATA 
3361 CGAGCCGGAA GCATAAAGTG TAAAGCCTGG GGTGCCTAAT GAGTGAGCTA ACTCACATTA 
3421 ATTGCGTTGC GCTCACTGCC CGCTTTCCAG TCGGGAAACC TGTCGTGCCA GCTGCATTAA 
3481 TGAATCGGCC AACGCGCGGG GAGAGGCGGT TTGCGTATTG GGCGCTCTTC CGCTTCCTCG 
3541 CTCACTGACT CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG 
3601 GCGGTAATAC GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT GTGAGCAAAA 
3661 GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CCATAGGCTC 
3721 CGCCCCCCTG ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA 
3781 GGACTATAAA GATACCAGGC GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG 
3841 ACCCTGCCGC TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT 
3901 CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT 
3961 GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG 
4021 TCCAACCCGG TAAGACACGA CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC 
4081 AGAGCGAGGT ATGTAGGCGG TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC 
4141 ACTAGAAGAA CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA 
4201 GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC 
4261 AAGCAGCAGA TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG 
4321 GGGTCTGACG CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT GAGATTATCA 
4381 AAAAGGATCT TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT 
4441 ATATATGAGT AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA 
4501 GCGATCTGTC TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA GATAACTACG 
4561 ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA TACCGCGAGA CCCACGCTCA 
4621 CCGGCTCCAG ATTTATCAGC AATAAACCAG CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT 
4681 CCTGCAACTT TATCCGCCTC CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT 
4741 AGTTCGCCAG TTAATAGTTT GCGCAACGTT GTTGCCATTG CTACAGGCAT CGTGGTGTCA 
4801 CGCTCGTCGT TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG GCGAGTTACA 
4861 TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA 
4921 AGTAAGTTGG CCGCAGTGTT ATCACTCATG GTTATGGCAG CACTGCATAA TTCTCTTACT 
4981 GTCATGCCAT CCGTAAGATG CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA 
5041 GAATAGTGTA TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAATACGGGA TAATACCGCG 
5101 CCACATAGCA GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG GCGAAAACTC 
5161 TCAAGGATCT TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC ACCCAACTGA 
5221 TCTTCAGCAT CTTTTACTTT CACCAGCGTT TCTGGGTGAG CAAAAACAGG AAGGCAAAAT 
5281 GCCGCAAAAA AGGGAATAAG GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT 
5341 CAATATTATT GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT 
5401 ATTTAGAAAA ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAC 
5461 GTCTAAGAAA CCATTATTAT CATGACATTA ACCTATAAAA ATAGGCGTAT CACGAGGCCC 
5521 TTTCGTCTCG CGCGTTTCGG TGATGACGGT GAAAACCTCT GACACATGCA GCTCCCGGAG 
5581 ACGGTCACAG CTTGTCTGTA AGCGGATGCC GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA 
5641 GCGGGTGTTG GCGGGTGTCG GGGCTGGCTT AACTATGCGG CATCAGAGCA GATTGTACTG 
5701 AGAGTGCACC ATATGCGGTG TGAAATACCG CACAGATGCG TAAGGAGAAA ATACCGCATC 
5761 AGGCGCCATT CGCCATTCAG GCTGCGCAAC TGTTGGGAAG GGCGATCGGT GCGGGCCTCT 
5821 TCGCTATTAC GCCAGCTGGC GAAAGGGGGA TGTGCTGCAA GGCGATTAAG TTGGGTAACG 
5881 CCAGGGTTTT CCCAGTCACG ACGTTGTAAA ACGACGGCCA GTGCCAAGCT TATG