RNAi-Ready pSIREN-RetroQ

RNAi-Ready pSIREN-RetroQ is a self-inactivating retroviral expression vector designed to express a small hairpin RNA (shRNA) using the human U6 promoter (PU6; RNA Pol III-dependent). RNAi-Ready pSIREN-RetroQ is provided as a linearized vector digested with BamH I and EcoR I. It is used for targeted gene silencing when an oligonuceotide encoding an appropriate shRNA is ligated into the vector. You can transfect your pSIREN-RetroQ construct as a plasmid expression vector, or—upon transfection into a packaging cell line—this vector can transiently express, or integrate and stably express a viral genomic transcript containing the human U6 promoter and the shRNA. The vector contains a puromycin resistance gene for the selection of stable transfectants.

This retroviral vector is optimized to eliminate promoter interference through self-inactivation. The hybrid 5' LTR consists of the cytomegalovirus (CMV) type I enhancer and the mouse sarcoma virus (MSV) promoter. This construct drives high levels of transcription in HEK 293-based packaging cell lines due, in part, to the presence of adenoviral E1A (1–4) in these cells. The self-inactivating feature of the vector is provided by a deletion in the 3' LTR enhancer region (U3). During reverse transcription of the retroviral RNA, the inactivated 3' LTR is copied and replaces the 5' LTR, resulting in inactivation of the 5' LTR CMV enhancer sequences. This may reduce the phenomenon known as promoter interference (5) and allow more efficient expression.

Also included in the viral genomic transcript are the necessary viral RNA processing elements including the LTRs, packaging signal (Psi+), and tRNA primer binding site. RNAi-Ready pSIREN-RetroQ also contains a bacterial origin of replication and E. coli Ampr gene for propagation and selection in bacteria.

载体应用

RNAi-Ready pSIREN-RetroQ is used for targeted gene silencing when an oligonuceotide encoding an appropriate shRNA is inserted. To construct recombinant pSIREN-RetroQ, first design, generate, and anneal complementary shRNA oligonucleotides using the protocols in the Knockout RNAi Systems User Manual (PT3739-1). The annealed oligonucleotide should contain 5'-BamH I and 3'-EcoR I sites. Then ligate the annealed oligonucleotide into RNAi-Ready pSIREN-RetroQ.

Your pSIREN-RetroQ construct can be transfected as a plasmid expression vector to screen for functional shRNA oligonucleotides. For gene silencing experiments using viral delivery, transfect the pSIREN-RetroQ construct into a packaging cell line (such as the RetroPack? PT67 Cell Line (Cat. No. 631510), AmphoPack?-293 Cell Line, EcoPack2?-293 Cell Line, or Pantropic System). RNA from the vector is packaged into infectious retroviral particles. These infectious particles are replication-incompetent since pSIREN-RetroQ lacks structural genes (gag, pol, and env) necessary for particle formation and replication; however, these genes are stably integrated as part of the packaging cell genome. These retroviral particles can infect a wide range of target cells and transmit the shRNA 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 chances of producing replication-competent virus due to recombination events during cell proliferation.

0001 TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC 
0061 CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC 
0121 GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA 
0181 TGCCAAGTAC GCCCCCTATT GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC 
0241 AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT CTACGTATTA GTCATCGCTA 
0301 TTACCATGGT GATGCGGTTT TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC 
0361 GGGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGG CACCAAAATC 
0421 AACGGGACTT TCCAAAATGT CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC 
0481 GTGTACGGTG GGAGGTCTAT ATAAGCAGAG CTCAATAAAA GAGCCCACAA CCCCTCACTC 
0541 GGCGCGCCAG TCTTCCGATA GACTGCGTCG CCCGGGTACC CGTATTCCCA ATAAAGCCTC 
0601 TTGCTGTTTG CATCCGAATC GTGGTCTCGC TGTTCCTTGG GAGGGTCTCC TCTGAGTGAT 
0661 TGACTACCCA CGACGGGGGT CTTTCATTTG GGGGCTCGTC CGGGATTTGG AGACCCCTGC 
0721 CCAGGGACCA CCGACCCACC ACCGGGAGGT AAGCTGGCCA GCAACTTATC TGTGTCTGTC 
0781 CGATTGTCTA GTGTCTATGT TTGATGTTAT GCGCCTGCGT CTGTACTAGT TAGCTAACTA 
0841 GCTCTGTATC TGGCGGACCC GTGGTGGAAC TGACGAGTTC TGAACACCCG GCCGCAACCC 
0901 TGGGAGACGT CCCAGGGACT TTGGGGGCCG TTTTTGTGGC CCGACCTGAG GAAGGGAGTC 
0961 GATGTGGAAT CCGACCCCGT CAGGATATGT GGTTCTGGTA GGAGACGAGA ACCTAAAACA 
1021 GTTCCCGCCT CCGTCTGAAT TTTTGCTTTC GGTTTGGAAC CGAAGCCGCG CGTCTTGTCT 
1081 GCTGCAGCGC TGCAGCATCG TTCTGTGTTG TCTCTGTCTG ACTGTGTTTC TGTATTTGTC 
1141 TGAAAATTAG GGCCAGACTG TTACCACTCC CTTAAGTTTG ACCTTAGGTC ACTGGAAAGA 
1201 TGTCGAGCGG ATCGCTCACA ACCAGTCGGT AGATGTCAAG AAGAGACGTT GGGTTACCTT 
1261 CTGCTCTGCA GAATGGCCAA CCTTTAACGT CGGATGGCCG CGAGACGGCA CCTTTAACCG 
1321 AGACCTCATC ACCCAGGTTA AGATCAAGGT CTTTTCACCT GGCCCGCATG GACACCCAGA 
1381 CCAGGTCCCC TACATCGTGA CCTGGGAAGC CTTGGCTTTT GACCCCCCTC CCTGGGTCAA 
1441 GCCCTTTGTA CACCCTAAGC CTCCGCCTCC TCTTCCTCCA TCCGCCCCGT CTCTCCCCCT 
1501 TGAACCTCCT CGTTCGACCC CGCCTCGATC CTCCCTTTAT CCAGCCCTCA CTCCTTCTCT 
1561 AGGCGCCGGA ATTGAAGATC TGGGCAGGAA GAGGGCCTAT TTCCCATGAT TCCTTCATAT 
1621 TTGCATATAC GATACAAGGC TGTTAGAGAG ATAATTAGAA TTAATTTGAC TGTAAACACA 
1681 AAGATATTAG TACAAAATAC GTGACGTAGA AAGTAATAAT TTCTTGGGTA GTTTGCAGTT 
1741 TTAAAATTAT GTTTTAAAAT GGACTATCAT ATGCTTACCG TAACTTGAAA GTATTTCGAT 
1801 TTCTTGGCTT TATATATCTT GTGGAAAGGA CGAGGATCCG GACAAGCTTC GAATTCTACC 
1861 GGGTAGGGGA GGCGCTTTTC CCAAGGCAGT CTGGAGCATG CGCTTTAGCA GCCCCGCTGG 
1921 GCACTTGGCG CTACACAAGT GGCCTCTGGC CTCGCACACA TTCCACATCC ACCGGTAGGC 
1981 GCCAACCGGC TCCGTTCTTT GGTGGCCCCT TCGCGCCACC TTCTACTCCT CCCCTAGTCA 
2041 GGAAGTTCCC CCCCGCCCCG CAGCTCGCGT CGTGCAGGAC GTGACAAATG GAAGTAGCAC 
2101 GTCTCACTAG TCTCGTGCAG ATGGACAGCA CCGCTGAGCA ATGGAAGCGG GTAGGCCTTT 
2161 GGGGCAGCGG CCAATAGCAG CTTTGCTCCT TCGCTTTCTG GGCTCAGAGG CTGGGAAGGG 
2221 GTGGGTCCGG GGGCGGGCTC AGGGGCGGGC TCAGGGGCGG GGCGGGCGCC CGAAGGTCCT 
2281 CCGGAGGCCC GGCATTCTGC ACGCTTCAAA AGCGCACGTC TGCCGCGCTG TTCTCCTCTT 
2341 CCTCATCTCC GGGCCTTTCG ACCTGCAGCC CAAGCTTACC ATGACCGAGT ACAAGCCCAC 
2401 GGTGCGCCTC GCCACCCGCG ACGACGTCCC CAGGGCCGTA CGCACCCTCG CCGCCGCGTT 
2461 CGCCGACTAC CCCGCCACGC GCCACACCGT CGATCCGGAC CGCCACATCG AGCGGGTCAC 
2521 CGAGCTGCAA GAACTCTTCC TCACGCGCGT CGGGCTCGAC ATCGGCAAGG TGTGGGTCGC 
2581 GGACGACGGC GCCGCGGTGG CGGTCTGGAC CACGCCGGAG AGCGTCGAAG CGGGGGCGGT 
2641 GTTCGCCGAG ATCGGCCCGC GCATGGCCGA GTTGAGCGGT TCCCGGCTGG CCGCGCAGCA 
2701 ACAGATGGAA GGCCTCCTGG CGCCGCACCG GCCCAAGGAG CCCGCGTGGT TCCTGGCCAC 
2761 CGTCGGCGTC TCGCCCGACC ACCAGGGCAA GGGTCTGGGC AGCGCCGTCG TGCTCCCCGG 
2821 AGTGGAGGCG GCCGAGCGCG CCGGGGTGCC CGCCTTCCTG GAGACCTCCG CGCCCCGCAA 
2881 CCTCCCCTTC TACGAGCGGC TCGGCTTCAC CGTCACCGCC GACGTCGAGG TGCCCGAAGG 
2941 ACCGCGCACC TGGTGCATGA CCCGCAAGCC CGGTGCCTGA CGCCCGCCCC ACGACCCGCA 
3001 GCGCCCGACC GAAAGGAGCG CACGACCCCA TGCATCGGCG TCTCGAGATA TCAGTGGTCC 
3061 AGGCTCTAGT TTTGACTCAA CAATATCACC AGCTGAAGCC TATAGAGTAC GAGCCATAGA 
3121 TAAAATAAAA GATTTTATTT AGTCTCCAGA AAAAGGGGGG AATGAAAGAC CCCACCTGTA 
3181 GGTTTGGCAA GCTAGCTTAA GTAACGCCAT TTTGCAAGGC ATGGAAAAAT ACATAACTGA 
3241 GAATAGAGAA GTTCAGATCA AGGTCAGGAA CAGATGGAAC AGGGTCGACC CTAGAGAACC 
3301 ATCAGATGTT TCCAGGGTGC CCCAAGGACC TGAAATGACC CTGTGCCTTA TTTGAACTAA 
3361 CCAATCAGTT CGCTTCTCGC TTCTGTTCGC GCGCTTCTGC TCCCCGAGCT CAATAAAAGA 
3421 GCCCACAACC CCTCACTCGG GGCGCCAGTC CTCCGATTGA CTGAGTCGCC CGGGTACCCG 
3481 TGTATCCAAT AAACCCTCTT GCAGTTGCAT CCGACTTGTG GTCTCGCTGT TCCTTGGGAG 
3541 GGTCTCCTCT GAGTGATTGA CTACCCGTCA GCGGGGGTCT TTCATTTGGG GGCTCGTCCG 
3601 GGATCGGGAG ACCCCTGCCC AGGGACCACC GACCCACCAC CGGGAGGTAA GCTGGCTGCC 
3661 TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA 
3721 CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG 
3781 TTGGCGGGTG TCGGGGCGCA GCCATGACCC AGTCACGTAG CGATAGCGGA GTGTAGATCC 
3841 GGCTGTGGAA TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA 
3901 GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCAGGTG TGGAAAGTCC CCAGGCTCCC 
3961 CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCATA GTCCCGCCCC 
4021 TAACTCCGCC CATCCCGCCC CTAACTCCGC CCAGTTCCGC CCATTCTCCG CCCCATGGCT 
4081 GACTAATTTT TTTTATTTAT GCAGAGGCCG AGGCCGCCTC GGCCTCTGAG CTATTCCAGA 
4141 AGTAGTGAGG AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA AAAGCTTACT GGCTTAACTA 
4201 TGCGGCATCA GAGCAGATTG TACTGAGAGT GCACCATATG CGGTGTGAAA TACCGCACAG 
4261 ATGCGTAAGG AGAAAATACC GCATCAGGCG CTCTTCCGCT TCCTCGCTCA CTGACTCGCT 
4321 GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG TAATACGGTT 
4381 ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC AGCAAAAGGC 
4441 CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA 
4501 GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC TATAAAGATA 
4561 CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC 
4621 CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA GCTCACGCTG 
4681 TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC 
4741 CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG 
4801 ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT 
4861 AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA GAAGGACAGT 
4921 ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG 
4981 ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC 
5041 GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA 
5101 GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC 
5161 CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT ATGAGTAAAC 
5221 TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA TCTGTCTATT 
5281 TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT 
5341 ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT 
5401 ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG CAACTTTATC 
5461 CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA 
5521 TAGTTTGCGC AACGTTGTTG CCATTGCTGC AGGCATCGTG GTGTCACGCT CGTCGTTTGG 
5581 TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT CCCCCATGTT 
5641 GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGC 
5701 AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA TGCCATCCGT 
5761 AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT AGTGTATGCG 
5821 GCGACCGAGT TGCTCTTGCC CGGCGTCAAC ACGGGATAAT ACCGCGCCAC ATAGCAGAAC 
5881 TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC 
5941 GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT 
6001 TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG 
6061 AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT ATTATTGAAG 
6121 CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA 
6181 ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTCT AAGAAACCAT 
6241 TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG AGGCCCTTTC GTCTTCAAGA 
6301 ATTAGCTTGG CCATTGCATA CGTTGTATCC ATATCATAAT ATGTACATTT ATATTGGCTC 
6361 ATGTCCAACA TTACCGCCAT GTTGACATTG ATTATTGACT AGTTATTAAT AGTAATCAAT 
6421 TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGT