描述

pIREShyg3 contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RNA (1–3). After selection with hygromycin B, nearly all surviving colonies will stably express the gene of interest, thus decreasing the need to screen large numbers of colonies to find functional clones. To select for cells that express high levels of the gene of interest, the selective pressure for antibiotic resistance was increased by shifting the hygromycin B phosphotransferase gene downstream to a less optimal position for translation as directed by the IRES sequence (3). By decreasing the level of expression of the antibiotic resistance marker, the selective pressure on the entire expression cassette is increased, resulting in selection for cells that express the entire transcript, including the gene of interest, at high levels. The expression cassette of pIREShyg3 contains the human cytomegalovirus (CMV) major immediate early promoter/enhancer followed by a multiple cloning site (MCS) that precedes stop codons in all three reading frames, a synthetic intron known to enhance the stability of the mRNA (4), the ECMV IRES followed by the hygromycin B phosphotransferase gene, and the polyadenylation signal from SV40. Ribosomes can enter the bicistronic mRNA at the 5' end to translate the gene of interest and at the ECMV IRES to translate the antibiotic resistance marker. ?

應(yīng)用

When using the pIREShyg3 Vector, the antibiotic exerts selective pressure on the entire expression cassette; thus, a high dose of antibiotic will select for cells expressing a high level of the gene of interest. This selective pressure also ensures that the expression of the gene of interest will be stable over time in culture. Unless your expression experiments require a pure population of cells, you can use the pool of cells surviving selection instead of isolating and characterizing clonal cell lines. We recommend selecting mammalian cultures in 250–600 μg/ml Hygromycin B (Cat. No. 631309) depending on the cell line. Be sure to establish a kill curve for each cell line and each lot of Hygromycin B to determine optimal selection concentration.

0001 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTCGACTCT CAGTACAATC TGCTCTGATG 
0061 CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG 
0121 CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA CAATTGCATG AAGAATCTGC 
0181 TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT 
0241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA 
0301 TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC 
0361 CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC 
0421 ATTGACGTCA ATGGGTGGAC TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT 
0481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT 
0541 ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA 
0601 TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG 
0661 ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC 
0721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG 
0781 GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA 
0841 CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG GGAGACCCAA GCTTGGTACC 
0901 GAGCTCGGAT CTGTACAGGC CTTAAGCCGG CGCGCTAGCG CTTCGAAGGC GCCCGGGATC 
0961 CGATATCGTA CGTTAACATA GATAACTGAT CCAGTGTGCT GGAATTAATT CGCTGTCTGC 
1021 GAGGGCCAGC TGTTGGGGTG AGTACTCCCT CTCAAAAGCG GGCATGACTT CTGCGCTAAG 
1081 ATTGTCAGTT TCCAAAAACG AGGAGGATTT GATATTCACC TGGCCCGCGG TGATGCCTTT 
1141 GAGGGTGGCC GCGTCCATCT GGTCAGAAAA GACAATCTTT TTGTTGTCAA GCTTGAGGTG 
1201 TGGCAGGCTT GAGATCTGGC CATACACTTG AGTGACAATG ACATCCACTT TGCCTTTCTC 
1261 TCCACAGGTG TCCACTCCCA GGTCCAACTG CAGGTCGAGC ATGCATCTAG GGCGGCCGCA 
1321 CTAGAGGAAT TCCGCCCCTC TCCCTCCCCC CCCCCTAACG TTACTGGCCG AAGCCGCTTG 
1381 GAATAAGGCC GGTGTGTGTT TGTCTATATG TTATTTTCCA CCATATTGCC GTCTTTTGGC 
1441 AATGTGAGGG CCCGGAAACC TGGCCCTGTC TTCTTGACGA GCATTCCTAG GGGTCTTTCC 
1501 CCTCTCGCCA AAGGAATGCA AGGTCTGTTG AATGTCGTGA AGGAAGCAGT TCCTCTGGAA 
1561 GCTTCTTGAA GACAAACAAC GTCTGTAGCG ACCCTTTGCA GGCAGCGGAA CCCCCCACCT 
1621 GGCGACAGGT GCCTCTGCGG CCAAAAGCCA CGTGTATAAG ATACACCTGC AAAGGCGGCA 
1681 CAACCCCAGT GCCACGTTGT GAGTTGGATA GTTGTGGAAA GAGTCAAATG GCTCTCCTCA 
1741 AGCGTAGTCA ACAAGGGGCT GAAGGATGCC CAGAAGGTAC CCCATTGTAT GGGAATCTGA 
1801 TCTGGGGCCT CGGTGCACAT GCTTTACATG TGTTTAGTCG AGGTTAAAAA AACGTCTAGG 
1861 CCCCCCGAAC CACGGGGACG TGGTTTTCCT TTGAAAAACA CGATGATAAG CTTGCCACAA 
1921 CCCGTACCAA AGATGGATAG ATCCGGAAAG CCTGAACTCA CCGCGACGTC TGTCGAGAAG 
1981 TTTCTGATCG AAAAGTTCGA CAGCGTCTCC GACCTGATGC AGCTCTCGGA GGGCGAAGAA 
2041 TCTCGTGCTT TCAGCTTCGA TGTAGGAGGG CGTGGATATG TCCTGCGGGT AAATAGCTGC 
2101 GCCGATGGTT TCTACAAAGA TCGTTATGTT TATCGGCACT TTGCATCGGC CGCGCTCCCG 
2161 ATTCCGGAAG TGCTTGACAT TGGGGAATTC AGCGAGAGCC TGACCTATTG CATCTCCCGC 
2221 CGTGCACAGG GTGTCACGTT GCAAGACCTG CCTGAAACCG AACTGCCCGC TGTTCTGCAG 
2281 CCGGTCGCGG AGGCCATGGA TGCGATCGCT GCGGCCGATC TTAGCCAGAC GAGCGGGTTC 
2341 GGCCCATTCG GACCGCAAGG AATCGGTCAA TACACTACAT GGCGTGATTT CATATGCGCG 
2401 ATTGCTGATC CCCATGTGTA TCACTGGCAA ACTGTGATGG ACGACACCGT CAGTGCGTCC 
2461 GTCGCGCAGG CTCTCGATGA GCTGATGCTT TGGGCCGAGG ACTGCCCCGA AGTCCGGCAC 
2521 CTCGTGCACG CGGATTTCGG CTCCAACAAT GTCCTGACGG ACAATGGCCG CATAACAGCG 
2581 GTCATTGACT GGAGCGAGGC GATGTTCGGG GATTCCCAAT ACGAGGTCGC CAACATCTTC 
2641 TTCTGGAGGC CGTGGTTGGC TTGTATGGAG CAGCAGACGC GCTACTTCGA GCGGAGGCAT 
2701 CCGGAGCTTG CAGGATCGCC GCGGCTCCGG GCGTATATGC TCCGCATTGG TCTTGACCAA 
2761 CTCTATCAGA GCTTGGTTGA CGGCAATTTC GATGATGCAG CTTGGGCGCA GGGTCGATGC 
2821 GACGCAATCG TCCGATCCGG AGCCGGGACT GTCGGGCGTA CACAAATCGC CCGCAGAAGC 
2881 GCGGCCGTCT GGACCGATGG CTGTGTAGAA GTACTCGCCG ATAGTGGAAA CCGACGCCCC 
2941 AGCACTCGTC CGAGGGCAAA GGAATAGAGT AGATGCCGAC CGAACAAGAG CTGATTTCGA 
3001 GAACGCCTCA GCCAGCAACT CGCGCGAGCC TAGCAAGGCA AATGCGAGAG AACGGCCTTA 
3061 CGCTTGGTGG CACAGTTCTC GTCCACAGTT CGCTAAGCTC GCTCGGCTGG GTCGCGGGAG 
3121 GGCCGGTCGC AGTGATTCAG GCCCTTCTGG ATTGTGTTGG TCCCCAGGGC ACGATTGTCA 
3181 TGCCCACGCA CTCGGGTGAT CTGACTGATC CCGCAGATTG GAGATCGCCG CCCGTGCCTG 
3241 CCGATTGGGT GCAGATCTAG ATAACTGATC ATAATCAGCC ATACCACATT TGTAGAGGTT 
3301 TTACTTGCTT TAAAAAACCT CCCACACCTC CCCCTGAACC TGAAACATAA AATGAATGCA 
3361 ATTGTTGTTG TTAACTTGTT TATTGCAGCT TATAATGGTT ACAAATAAAG CAATAGCATC 
3421 ACAAATTTCA CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGTTT GTCCAAACTC 
3481 ATCAATGTAT CTTAACGCTC GAGTGCATTC TAGTTGTGGT TTGTCCAAAC TCATCAATGT 
3541 ATCTTATCAT GTCTGTATAC CGTCGACCTC TAGCTAGAGC TTGGCGTAAT CATGGTCATA 
3601 GCTGTTTCCT GTGTGAAATT GTTATCCGCT CACAATTCCA CACAACATAC GAGCCGGAAG 
3661 CATAAAGTGT AAAGCCTGGG GTGCCTAATG AGTGAGCTAA CTCACATTAA TTGCGTTGCG 
3721 CTCACTGCCC GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT GAATCGGCCA 
3781 ACGCGCGGGG AGAGGCGGTT TGCGTATTGG GCGCTCTTCC GCTTCCTCGC TCACTGACTC 
3841 GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG 
3901 GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA 
3961 GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA 
4021 CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG 
4081 ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT 
4141 TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC AATGCTCACG 
4201 CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC 
4261 CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT 
4321 AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA 
4381 TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC 
4441 AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC 
4501 TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT 
4561 TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC 
4621 TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT 
4681 CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA 
4741 AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT 
4801 ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA TACGGGAGGG 
4861 CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC CCACGCTCAC CGGCTCCAGA 
4921 TTTATCAGCA ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT 
4981 ATCCGCCTCC ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT 
5041 TAATAGTTTG CGCAACGTTG TTGCCATTGC TACAGGCATC GTGGTGTCAC GCTCGTCGTT 
5101 TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT 
5161 GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA GTAAGTTGGC 
5221 CGCAGTGTTA TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG TCATGCCATC 
5281 CGTAAGATGC TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT 
5341 GCGGCGACCG AGTTGCTCTT GCCCGGCGTC AATACGGGAT AATACCGCGC CACATAGCAG 
5401 AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT 
5461 ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT CTTCAGCATC 
5521 TTTTACTTTC ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG CCGCAAAAAA 
5581 GGGAATAAGG GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG 
5641 AAGCATTTAT CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA 
5701 TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG TC