RNAi-Ready pSIREN-RetroQ-ZsGreen

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 pSIRENRetroQ- 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 GAATTCTAGT TATTAATAGT AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA 
0061 GTTCCGCGTT ACATAACTTA CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG 
0121 CCCATTGACG TCAATAATGA CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG 
0181 ACGTCAATGG GTGGAGTATT TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA 
0241 TATGCCAAGT ACGCCCCCTA TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC 
0301 CCAGTACATG ACCTTATGGG ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC 
0361 TATTACCATG GTGATGCGGT TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC 
0421 ACGGGGATTT CCAAGTCTCC ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA 
0481 TCAACGGGAC TTTCCAAAAT GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG 
0541 GCGTGTACGG TGGGAGGTCT ATATAAGCAG AGCTGGTTTA GTGAACCGTC AGATCCGCTA 
0601 GCGCTACCGG TCGCCACCAT GGCCCAGTCC AAGCACGGCC TGACCAAGGA GATGACCATG 
0661 AAGTACCGCA TGGAGGGCTG CGTGGACGGC CACAAGTTCG TGATCACCGG CGAGGGCATC 
0721 GGCTACCCCT TCAAGGGCAA GCAGGCCATC AACCTGTGCG TGGTGGAGGG CGGCCCCTTG 
0781 CCCTTCGCCG AGGACATCTT GTCCGCCGCC TTCATGTACG GCAACCGCGT GTTCACCGAG 
0841 TACCCCCAGG ACATCGTCGA CTACTTCAAG AACTCCTGCC CCGCCGGCTA CACCTGGGAC 
0901 CGCTCCTTCC TGTTCGAGGA CGGCGCCGTG TGCATCTGCA ACGCCGACAT CACCGTGAGC 
0961 GTGGAGGAGA ACTGCATGTA CCACGAGTCC AAGTTCTACG GCGTGAACTT CCCCGCCGAC 
1021 GGCCCCGTGA TGAAGAAGAT GACCGACAAC TGGGAGCCCT CCTGCGAGAA GATCATCCCC 
1081 GTGCCCAAGC AGGGCATCTT GAAGGGCGAC GTGAGCATGT ACCTGCTGCT GAAGGACGGT 
1141 GGCCGCTTGC GCTGCCAGTT CGACACCGTG TACAAGGCCA AGTCCGTGCC CCGCAAGATG 
1201 CCCGACTGGC ACTTCATCCA GCACAAGCTG ACCCGCGAGG ACCGCAGCGA CGCCAAGAAC 
1261 CAGAAGTGGC ACCTGACCGA GCACGCCATC GCCTCCGGCT CCGCCTTGCC CTAACTCGAG 
1321 ATATCAGTGG TCCAGGCTCT AGTTTTGACT CAACAATATC ACCAGCTGAA GCCTATAGAG 
1381 TACGAGCCAT AGATAAAATA AAAGATTTTA TTTAGTCTCC AGAAAAAGGG GGGAATGAAA 
1441 GACCCCACCT GTAGGTTTGG CAAGCTAGCT TAAGTAACGC CATTTTGCAA GGCATGGAAA 
1501 AATACATAAC TGAGAATAGA GAAGTTCAGA TCAAGGTCAG GAACAGATGG AACAGGGTCG 
1561 ACCCTAGAGA ACCATCAGAT GTTTCCAGGG TGCCCCAAGG ACCTGAAATG ACCCTGTGCC 
1621 TTATTTGAAC TAACCAATCA GTTCGCTTCT CGCTTCTGTT CGCGCGCTTC TGCTCCCCGA 
1681 GCTCAATAAA AGAGCCCACA ACCCCTCACT CGGGGCGCCA GTCCTCCGAT TGACTGAGTC 
1741 GCCCGGGTAC CCGTGTATCC AATAAACCCT CTTGCAGTTG CATCCGACTT GTGGTCTCGC 
1801 TGTTCCTTGG GAGGGTCTCC TCTGAGTGAT TGACTACCCG TCAGCGGGGG TCTTTCATTT 
1861 GGGGGCTCGT CCGGGATCGG GAGACCCCTG CCCAGGGACC ACCGACCCAC CACCGGGAGG 
1921 TAAGCTGGCT GCCTCGCGCG TTTCGGTGAT GACGGTGAAA ACCTCTGACA CATGCAGCTC 
1981 CCGGAGACGG TCACAGCTTG TCTGTAAGCG GATGCCGGGA GCAGACAAGC CCGTCAGGGC 
2041 GCGTCAGCGG GTGTTGGCGG GTGTCGGGGC GCAGCCATGA CCCAGTCACG TAGCGATAGC 
2101 GGAGTGTAGA TCCGGCTGTG GAATGTGTGT CAGTTAGGGT GTGGAAAGTC CCCAGGCTCC 
2161 CCAGCAGGCA GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAG GTGTGGAAAG 
2221 TCCCCAGGCT CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC 
2281 ATAGTCCCGC CCCTAACTCC GCCCATCCCG CCCCTAACTC CGCCCAGTTC CGCCCATTCT 
2341 CCGCCCCATG GCTGACTAAT TTTTTTTATT TATGCAGAGG CCGAGGCCGC CTCGGCCTCT 
2401 GAGCTATTCC AGAAGTAGTG AGGAGGCTTT TTTGGAGGCC TAGGCTTTTG CAAAAAGCTT 
2461 ACTGGCTTAA CTATGCGGCA TCAGAGCAGA TTGTACTGAG AGTGCACCAT ATGCGGTGTG 
2521 AAATACCGCA CAGATGCGTA AGGAGAAAAT ACCGCATCAG GCGCTCTTCC GCTTCCTCGC 
2581 TCACTGACTC GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG 
2641 CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG 
2701 GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC 
2761 GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG 
2821 GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA 
2881 CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC 
2941 ATAGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG 
3001 TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT 
3061 CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA 
3121 GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA 
3181 CTAGAAGGAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG 
3241 TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA 
3301 AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG 
3361 GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA 
3421 AAAGGATCTT CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA 
3481 TATATGAGTA AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG 
3541 CGATCTGTCT ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA 
3601 TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC CCACGCTCAC 
3661 CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC 
3721 CTGCAACTTT ATCCGCCTCC ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA 
3781 GTTCGCCAGT TAATAGTTTG CGCAACGTTG TTGCCATTGC TGCAGGCATC GTGGTGTCAC 
3841 GCTCGTCGTT TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT 
3901 GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA 
3961 GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG 
4021 TCATGCCATC CGTAAGATGC TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG 
4081 AATAGTGTAT GCGGCGACCG AGTTGCTCTT GCCCGGCGTC AACACGGGAT AATACCGCGC 
4141 CACATAGCAG AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT 
4201 CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT 
4261 CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG 
4321 CCGCAAAAAA GGGAATAAGG GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC 
4381 AATATTATTG AAGCATTTAT CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA 
4441 TTTAGAAAAA TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG 
4501 TCTAAGAAAC CATTATTATC ATGACATTAA CCTATAAAAA TAGGCGTATC ACGAGGCCCT 
4561 TTCGTCTTCA AGAATTAGCT TGGCCATTGC ATACGTTGTA TCCATATCAT AATATGTACA 
4621 TTTATATTGG CTCATGTCCA ACATTACCGC CATGTTGACA TTGATTATTG ACTAGTTATT 
4681 AATAGTAATC AATTACGGGG TCATTAGTTC ATAGCCCATA TATGGAGTTC CGCGTTACAT 
4741 AACTTACGGT AAATGGCCCG CCTGGCTGAC CGCCCAACGA CCCCCGCCCA TTGACGTCAA 
4801 TAATGACGTA TGTTCCCATA GTAACGCCAA TAGGGACTTT CCATTGACGT CAATGGGTGG 
4861 AGTATTTACG GTAAACTGCC CACTTGGCAG TACATCAAGT GTATCATATG CCAAGTACGC 
4921 CCCCTATTGA CGTCAATGAC GGTAAATGGC CCGCTCTGGC ATTATGCCCA GTACATGACC 
4981 TTATGGGACT TTCCTACTTG GCAGTACATC TACGTATTAG TCATCGCTAT TACCATGGTG 
5041 ATGCGGTTTT GGCAGTACAT CAATGGGCGT GGATAGCGGT TTGACTCACG GGGATTTCCA 
5101 AGTCTCCACC CCATTGACGT CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGACTTT 
5161 CCAAAATGTC GTAACAACTC CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG 
5221 GAGGTCTATA TAAGCAGAGC TCAATAAAAG AGCCCACAAC CCCTCACTCG GCGCGCCAGT 
5281 CTTCCGATAG ACTGCGTCGC CCGGGTACCC GTATTCCCAA TAAAGCCTCT TGCTGTTTGC 
5341 ATCCGAATCG TGGTCTCGCT GTTCCTTGGG AGGGTCTCCT CTGAGTGATT GACTACCCAC 
5401 GACGGGGGTC TTTCATTTGG GGGCTCGTCC GGGATTTGGA GACCCCTGCC CAGGGACCAC 
5461 CGACCCACCA CCGGGAGGTA AGCTGGCCAG CAACTTATCT GTGTCTGTCC GATTGTCTAG 
5521 TGTCTATGTT TGATGTTATG CGCCTGCGTC TGTACTAGTT AGCTAACTAG CTCTGTATCT 
5581 GGCGGACCCG TGGTGGAACT GACGAGTTCT GAACACCCGG CCGCAACCCT GGGAGACGTC 
5641 CCAGGGACTT TGGGGGCCGT TTTTGTGGCC CGACCTGAGG AAGGGAGTCG ATGTGGAATC 
5701 CGACCCCGTC AGGATATGTG GTTCTGGTAG GAGACGAGAA CCTAAAACAG TTCCCGCCTC 
5761 CGTCTGAATT TTTGCTTTCG GTTTGGAACC GAAGCCGCGC GTCTTGTCTG CTGCAGCGCT 
5821 GCAGCATCGT TCTGTGTTGT CTCTGTCTGA CTGTGTTTCT GTATTTGTCT GAAAATTAGG 
5881 GCCAGACTGT TACCACTCCC TTAAGTTTGA CCTTAGGTCA CTGGAAAGAT GTCGAGCGGA 
5941 TCGCTCACAA CCAGTCGGTA GATGTCAAGA AGAGACGTTG GGTTACCTTC TGCTCTGCAG 
6001 AATGGCCAAC CTTTAACGTC GGATGGCCGC GAGACGGCAC CTTTAACCGA GACCTCATCA 
6061 CCCAGGTTAA GATCAAGGTC TTTTCACCTG GCCCGCATGG ACACCCAGAC CAGGTCCCCT 
6121 ACATCGTGAC CTGGGAAGCC TTGGCTTTTG ACCCCCCTCC CTGGGTCAAG CCCTTTGTAC 
6181 ACCCTAAGCC TCCGCCTCCT CTTCCTCCAT CCGCCCCGTC TCTCCCCCTT GAACCTCCTC 
6241 GTTCGACCCC GCCTCGATCC TCCCTTTATC CAGCCCTCAC TCCTTCTCTA GGCGCCGGAA 
6301 TTGAAGATCT GGGCAGGAAG AGGGCCTATT TCCCATGATT CCTTCATATT TGCATATACG 
6361 ATACAAGGCT GTTAGAGAGA TAATTAGAAT TAATTTGACT GTAAACACAA AGATATTAGT 
6421 ACAAAATACG TGACGTAGAA AGTAATAATT TCTTGGGTAG TTTGCAGTTT TAAAATTATG 
6481 TTTTAAAATG GACTATCATA TGCTTACCGT AACTTGAAAG TATTTCGATT TCTTGGCTTT 
6541 ATATATCTTG TGGAAAGGAC GAGGATCC