RNAi-Ready pSIREN-RetroQ-ZsGreen1

RNAi-Ready pSIREN-RetroQ-ZsGreen1 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-ZsGreen1 is provided as a linearized vector digested with BamHI and EcoRI. It is used for targeted gene silencing when a ds DNA oligonucleotide encoding an appropriate shRNA is ligated into the vector. You can transfect your pSIREN-RetroQ-ZsGreen1 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.

In addition, this vector independently expresses a Zoanthus sp. green fluorescent protein (ZsGreen1; 1), which has been engineered for brighter fluorescence (excitation maximum = 496 nm; emission maximum = 506 nm). The ZsGreen1 gene is positioned just downstream of the immediate early promoter of cytomegalovirus (PCMV IE). As a result, cells transfected with this vector will express the green fluorescent protein constitutively.The ZsGreen1 fluorescent marker allows you to directly monitor the delivery efficiency of your gene silencing construct using either fluorescence microscopy or flow cytometry.

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 (2–5) 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 (6) and allow for 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-ZsGreen1 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-ZsGreen1 is used for targeted gene silencing when a ds DNA oligonucleotide encoding an appropriate shRNA is inserted. To construct recombinant pSIREN-RetroQ-ZsGreen1, 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'-BamHI and 3'-EcoRI overhangs. Then ligate the annealed oligonucleotide into RNAi-Ready pSIREN-RetroQ-ZsGreen1.

Your pSIREN-RetroQ-ZsGreen1 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-ZsGreen1 construct into a packaging cell line (see the Retroviral Gene Transfer and Expression User Manual, PT3132-1, for a list of packaging cell lines available from Clontech Laboratories, Inc.); RNA from the vector is packaged into infectious retroviral particles. 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 ZsGreen1 fluorescent marker in this vector allows direct monitoring of the delivery of your gene silencing construct. Use fluorescence microscopy or flow cytometry to easily detect or enrich for cells containing your recombinant shRNA vector.

0001 AATTCTAGTT ATTAATAGTA ATCAATTACG GGGTCATTAG TTCATAGCCC ATATATGGAG 
0061 TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT GACCGCCCAA CGACCCCCGC 
0121 CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGC CAATAGGGAC TTTCCATTGA 
0181 CGTCAATGGG TGGAGTATTT ACGGTAAACT GCCCACTTGG CAGTACATCA AGTGTATCAT 
0241 ATGCCAAGTA CGCCCCCTAT TGACGTCAAT GACGGTAAAT GGCCCGCCTG GCATTATGCC 
0301 CAGTACATGA CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTACGTATT AGTCATCGCT 
0361 ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC GTGGATAGCG GTTTGACTCA 
0421 CGGGGATTTC CAAGTCTCCA CCCCATTGAC GTCAATGGGA GTTTGTTTTG GCACCAAAAT 
0481 CAACGGGACT TTCCAAAATG TCGTAACAAC TCCGCCCCAT TGACGCAAAT GGGCGGTAGG 
0541 CGTGTACGGT GGGAGGTCTA TATAAGCAGA GCTGGTTTAG TGAACCGTCA GATCCGCTAG 
0601 CGCTACCGGT CGCCACCATG GCCCAGTCCA AGCACGGCCT GACCAAGGAG ATGACCATGA 
0661 AGTACCGCAT GGAGGGCTGC GTGGACGGCC ACAAGTTCGT GATCACCGGC GAGGGCATCG 
0721 GCTACCCCTT CAAGGGCAAG CAGGCCATCA ACCTGTGCGT GGTGGAGGGC GGCCCCTTGC 
0781 CCTTCGCCGA GGACATCTTG TCCGCCGCCT TCATGTACGG CAACCGCGTG TTCACCGAGT 
0841 ACCCCCAGGA CATCGTCGAC TACTTCAAGA ACTCCTGCCC CGCCGGCTAC ACCTGGGACC 
0901 GCTCCTTCCT GTTCGAGGAC GGCGCCGTGT GCATCTGCAA CGCCGACATC ACCGTGAGCG 
0961 TGGAGGAGAA CTGCATGTAC CACGAGTCCA AGTTCTACGG CGTGAACTTC CCCGCCGACG 
1021 GCCCCGTGAT GAAGAAGATG ACCGACAACT GGGAGCCCTC CTGCGAGAAG ATCATCCCCG 
1081 TGCCCAAGCA GGGCATCTTG AAGGGCGACG TGAGCATGTA CCTGCTGCTG AAGGACGGTG 
1141 GCCGCTTGCG CTGCCAGTTC GACACCGTGT ACAAGGCCAA GTCCGTGCCC CGCAAGATGC 
1201 CCGACTGGCA CTTCATCCAG CACAAGCTGA CCCGCGAGGA CCGCAGCGAC GCCAAGAACC 
1261 AGAAGTGGCA CCTGACCGAG CACGCCATCG CCTCCGGCTC CGCCTTGCCC TAACTCGAGA 
1321 TATCAGTGGT CCAGGCTCTA GTTTTGACTC AACAATATCA CCAGCTGAAG CCTATAGAGT 
1381 ACGAGCCATA GATAAAATAA AAGATTTTAT TTAGTCTCCA GAAAAAGGGG GGAATGAAAG 
1441 ACCCCACCTG TAGGTTTGGC AAGCTAGCTT AAGTAACGCC ATTTTGCAAG GCATGGAAAA 
1501 ATACATAACT GAGAATAGAG AAGTTCAGAT CAAGGTCAGG AACAGATGGA ACAGGGTCGA 
1561 CCCTAGAGAA CCATCAGATG TTTCCAGGGT GCCCCAAGGA CCTGAAATGA CCCTGTGCCT 
1621 TATTTGAACT AACCAATCAG TTCGCTTCTC GCTTCTGTTC GCGCGCTTCT GCTCCCCGAG 
1681 CTCAATAAAA GAGCCCACAA CCCCTCACTC GGGGCGCCAG TCCTCCGATT GACTGAGTCG 
1741 CCCGGGTACC CGTGTATCCA ATAAACCCTC TTGCAGTTGC ATCCGACTTG TGGTCTCGCT 
1801 GTTCCTTGGG AGGGTCTCCT CTGAGTGATT GACTACCCGT CAGCGGGGGT CTTTCATTTG 
1861 GGGGCTCGTC CGGGATCGGG AGACCCCTGC CCAGGGACCA CCGACCCACC ACCGGGAGGT 
1921 AAGCTGGCTG CCTCGCGCGT TTCGGTGATG ACGGTGAAAA CCTCTGACAC ATGCAGCTCC 
1981 CGGAGACGGT CACAGCTTGT CTGTAAGCGG ATGCCGGGAG CAGACAAGCC CGTCAGGGCG 
2041 CGTCAGCGGG TGTTGGCGGG TGTCGGGGCG CAGCCATGAC CCAGTCACGT AGCGATAGCG 
2101 GAGTGTAGAT CCGGCTGTGG AATGTGTGTC AGTTAGGGTG TGGAAAGTCC CCAGGCTCCC 
2161 CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCAGG TGTGGAAAGT 
2221 CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA 
2281 TAGTCCCGCC CCTAACTCCG CCCATCCCGC CCCTAACTCC GCCCAGTTCC GCCCATTCTC 
2341 CGCCCCATGG CTGACTAATT TTTTTTATTT ATGCAGAGGC CGAGGCCGCC TCGGCCTCTG 
2401 AGCTATTCCA GAAGTAGTGA GGAGGCTTTT TTGGAGGCCT AGGCTTTTGC AAAAAGCTTA 
2461 CTGGCTTAAC TATGCGGCAT CAGAGCAGAT TGTACTGAGA GTGCACCATA TGCGGTGTGA 
2521 AATACCGCAC AGATGCGTAA GGAGAAAATA CCGCATCAGG CGCTCTTCCG CTTCCTCGCT 
2581 CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGCTC ACTCAAAGGC 
2641 GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACATGT GAGCAAAAGG 
2701 CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG 
2761 CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACAGG 
2821 ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC 
2881 CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG CGCTTTCTCA 
2941 TAGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT 
3001 GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTATC GTCTTGAGTC 
3061 CAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGCAG 
3121 AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACT ACGGCTACAC 
3181 TAGAAGGACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTTCG GAAAAAGAGT 
3241 TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA 
3301 GCAGCAGATT ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTACGGG 
3361 GTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGA GATTATCAAA 
3421 AAGGATCTTC ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATCAA TCTAAAGTAT 
3481 ATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGCAC CTATCTCAGC 
3541 GATCTGTCTA TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTACGAT 
3601 ACGGGAGGGC TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGACC CACGCTCACC 
3661 GGCTCCAGAT TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC 
3721 TGCAACTTTA TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAGTAG 
3781 TTCGCCAGTT AATAGTTTGC GCAACGTTGT TGCCATTGCT GCAGGCATCG TGGTGTCACG 
3841 CTCGTCGTTT GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC GAGTTACATG 
3901 ATCCCCCATG TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAGAAG 
3961 TAAGTTGGCC GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT 
4021 CATGCCATCC GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA 
4081 ATAGTGTATG CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ACACGGGATA ATACCGCGCC 
4141 ACATAGCAGA ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACTCTC 
4201 AAGGATCTTA CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCAC CCAACTGATC 
4261 TTCAGCATCT TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC 
4321 CGCAAAAAAG GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACTCT TCCTTTTTCA 
4381 ATATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT 
4441 TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCTGACGT 
4501 CTAAGAAACC ATTATTATCA TGACATTAAC CTATAAAAAT AGGCGTATCA CGAGGCCCTT 
4561 TCGTCTTCAA GAATTAGCTT GGCCATTGCA TACGTTGTAT CCATATCATA ATATGTACAT 
4621 TTATATTGGC TCATGTCCAA CATTACCGCC ATGTTGACAT TGATTATTGA CTAGTTATTA 
4681 ATAGTAATCA ATTACGGGGT CATTAGTTCA TAGCCCATAT ATGGAGTTCC GCGTTACATA 
4741 ACTTACGGTA AATGGCCCGC CTGGCTGACC GCCCAACGAC CCCCGCCCAT TGACGTCAAT 
4801 AATGACGTAT GTTCCCATAG TAACGCCAAT AGGGACTTTC CATTGACGTC AATGGGTGGA 
4861 GTATTTACGG TAAACTGCCC ACTTGGCAGT ACATCAAGTG TATCATATGC CAAGTACGCC 
4921 CCCTATTGAC GTCAATGACG GTAAATGGCC CGCCTGGCAT TATGCCCAGT ACATGACCTT 
4981 ATGGGACTTT CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA CCATGGTGAT 
5041 GCGGTTTTGG CAGTACATCA ATGGGCGTGG ATAGCGGTTT GACTCACGGG GATTTCCAAG 
5101 TCTCCACCCC ATTGACGTCA ATGGGAGTTT GTTTTGGCAC CAAAATCAAC GGGACTTTCC 
5161 AAAATGTCGT AACAACTCCG CCCCATTGAC GCAAATGGGC GGTAGGCGTG TACGGTGGGA 
5221 GGTCTATATA AGCAGAGCTC AATAAAAGAG CCCACAACCC CTCACTCGGC GCGCCAGTCT 
5281 TCCGATAGAC TGCGTCGCCC GGGTACCCGT ATTCCCAATA AAGCCTCTTG CTGTTTGCAT 
5341 CCGAATCGTG GTCTCGCTGT TCCTTGGGAG GGTCTCCTCT GAGTGATTGA CTACCCACGA 
5401 CGGGGGTCTT TCATTTGGGG GCTCGTCCGG GATTTGGAGA CCCCTGCCCA GGGACCACCG 
5461 ACCCACCACC GGGAGGTAAG CTGGCCAGCA ACTTATCTGT GTCTGTCCGA TTGTCTAGTG 
5521 TCTATGTTTG ATGTTATGCG CCTGCGTCTG TACTAGTTAG CTAACTAGCT CTGTATCTGG 
5581 CGGACCCGTG GTGGAACTGA CGAGTTCTGA ACACCCGGCC GCAACCCTGG GAGACGTCCC 
5641 AGGGACTTTG GGGGCCGTTT TTGTGGCCCG ACCTGAGGAA GGGAGTCGAT GTGGAATCCG 
5701 ACCCCGTCAG GATATGTGGT TCTGGTAGGA GACGAGAACC TAAAACAGTT CCCGCCTCCG 
5761 TCTGAATTTT TGCTTTCGGT TTGGAACCGA AGCCGCGCGT CTTGTCTGCT GCAGCGCTGC 
5821 AGCATCGTTC TGTGTTGTCT CTGTCTGACT GTGTTTCTGT ATTTGTCTGA AAATTAGGGC 
5881 CAGACTGTTA CCACTCCCTT AAGTTTGACC TTAGGTCACT GGAAAGATGT CGAGCGGATC 
5941 GCTCACAACC AGTCGGTAGA TGTCAAGAAG AGACGTTGGG TTACCTTCTG CTCTGCAGAA 
6001 TGGCCAACCT TTAACGTCGG ATGGCCGCGA GACGGCACCT TTAACCGAGA CCTCATCACC 
6061 CAGGTTAAGA TCAAGGTCTT TTCACCTGGC CCGCATGGAC ACCCAGACCA GGTCCCCTAC 
6121 ATCGTGACCT GGGAAGCCTT GGCTTTTGAC CCCCCTCCCT GGGTCAAGCC CTTTGTACAC 
6181 CCTAAGCCTC CGCCTCCTCT TCCTCCATCC GCCCCGTCTC TCCCCCTTGA ACCTCCTCGT 
6241 TCGACCCCGC CTCGATCCTC CCTTTATCCA GCCCTCACTC CTTCTCTAGG CGCCGGAATT 
6301 GAAGATCTGG GCAGGAAGAG GGCCTATTTC CCATGATTCC TTCATATTTG CATATACGAT 
6361 ACAAGGCTGT TAGAGAGATA ATTAGAATTA ATTTGACTGT AAACACAAAG ATATTAGTAC 
6421 AAAATACGTG ACGTAGAAAG TAATAATTTC TTGGGTAGTT TGCAGTTTTA AAATTATGTT 
6481 TTAAAATGGA CTATCATATG CTTACCGTAA CTTGAAAGTA TTTCGATTTC TTGGCTTTAT 
6541 ATATCTTGTG GAAAGGACGA GG