pIRESneo3 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 G418, 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 neomycin phosphotransferase gene downstream to a less optimal position for translation as directed by the IRES sequence (1). By decreasing the level of expression of the antibiotic resistance marker, the selective pressure on the entire expression cassette is increased, resulting in the selection of cells that express the entire transcript, including the gene of interest, at high levels.

The expression cassette of pIRESneo3 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 neomycin phosphotransferase (NPT II) 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. Use: When using the pIRESneo3 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 500–1,300 μg/ml G418 (Cat. No. 631307) depending on the cell line. Be sure to establish a kill curve for each cell line and each lot of G418 to determine optimal selection concentration.

載體應(yīng)用

When using the pIRESneo3 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 500–1,300 μg/ml G418 (Cat. No. 631307) depending on the cell line. Be sure to establish a kill curve for each cell line and each lot of G418 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 CGATATCTGC GGCCTAGCTA GCGCTTAAGG CCTGTTAACC GGTCGTACGT 
0961 CTCCGGATTC GAATTCGGAT CCGCGGCCGC ATAGATAACT GATCCAGTGT GCTGGAATTA 
1021 ATTCGCTGTC TGCGAGGGCC AGCTGTTGGG GTGAGTACTC CCTCTCAAAA GCGGGCATGA 
1081 CTTCTGCGCT AAGATTGTCA GTTTCCAAAA ACGAGGAGGA TTTGATATTC ACCTGGCCCG 
1141 CGGTGATGCC TTTGAGGGTG GCCGCGTCCA TCTGGTCAGA AAAGACAATC TTTTTGTTGT 
1201 CAAGCTTGAG GTGTGGCAGG CTTGAGATCT GGCCATACAC TTGAGTGACA ATGACATCCA 
1261 CTTTGCCTTT CTCTCCACAG GTGTCCACTC CCAGGTCCAA CTGCAGGTCG AGCATGCATC 
1321 TAGGGCGGCC AATTCCGCCC CTCTCCCTCC CCCCCCCCTA ACGTTACTGG CCGAAGCCGC 
1381 TTGGAATAAG GCCGGTGTGC GTTTGTCTAT ATGTTATTTT CCACCATATT GCCGTCTTTT 
1441 GGCAATGTGA GGGCCCGGAA ACCTGGCCCT GTCTTCTTGA CGAGCATTCC TAGGGGTCTT 
1501 TCCCCTCTCG CCAAAGGAAT GCAAGGTCTG TTGAATGTCG TGAAGGAAGC AGTTCCTCTG 
1561 GAAGCTTCTT GAAGACAAAC AACGTCTGTA GCGACCCTTT GCAGGCAGCG GAACCCCCCA 
1621 CCTGGCGACA GGTGCCTCTG CGGCCAAAAG CCACGTGTAT AAGATACACC TGCAAAGGCG 
1681 GCACAACCCC AGTGCCACGT TGTGAGTTGG ATAGTTGTGG AAAGAGTCAA ATGGCTCTCC 
1741 TCAAGCGTAT TCAACAAGGG GCTGAAGGAT GCCCAGAAGG TACCCCATTG TATGGGATCT 
1801 GATCTGGGGC CTCGGTGCAC ATGCTTTACA TGTGTTTAGT CGAGGTTAAA AAAACGTCTA 
1861 GGCCCCCCGA ACCACGGGGA CGTGGTTTTC CTTTGAAAAA CACGATGATA AGCTTGCCAC 
1921 AACCCGGGAT AATTCCTGCA GCCAATATGG GATCGGCCAT TGAACAAGAT GGATTGCACG 
1981 CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA 
2041 TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG 
2101 TCAAGACCGA CCTGTCCGGT GCCCTGAATG AACTGCAGGA CGAGGCAGCG CGGCTATCGT 
2161 GGCTGGCCAC GACGGGCGTT CCTTGCGCAG CTGTGCTCGA CGTTGTCACT GAAGCGGGAA 
2221 GGGACTGGCT GCTATTGGGC GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC 
2281 CTGCCGAGAA AGTATCCATC ATGGCTGATG CAATGCGGCG GCTGCATACG CTTGATCCGG 
2341 CTACCTGCCC ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT ACTCGGATGG 
2401 AAGCCGGTCT TGTCGATCAG GATGATCTGG ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG 
2461 AACTGTTCGC CAGGCTCAAG GCGCGCATGC CCGACGGCGA TGATCTCGTC GTGACCCATG 
2521 GCGATGCCTG CTTGCCGAAT ATCATGGTGG AAAATGGCCG CTTTTCTGGA TTCATCGACT 
2581 GTGGCCGGCT GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC CGTGATATTG 
2641 CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT ATCGCCGCTC 
2701 CCGATTCGCA GCGCATCGCC TTCTATCGCC TTCTTGACGA GTTCTTCTGA GGGGATCAAT 
2761 TCTCTAGATA ACTGATCATA ATCAGCCATA CCACATTTGT AGAGGTTTTA CTTGCTTTAA 
2821 AAAACCTCCC ACACCTCCCC CTGAACCTGA AACATAAAAT GAATGCAATT GTTGTTGTTA 
2881 ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA TAGCATCACA AATTTCACAA 
2941 ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC CAAACTCATC AATGTATCTT 
3001 AACGCGTCGA GTGCATTCTA GTTGTGGTTT GTCCAAACTC ATCAATGTAT CTTATCATGT 
3061 CTGTATACCG TCGACCTCTA GCTAGAGCTT GGCGTAATCA TGGTCATAGC TGTTTCCTGT 
3121 GTGAAATTGT TATCCGCTCA CAATTCCACA CAACATACGA GCCGGAAGCA TAAAGTGTAA 
3181 AGCCTGGGGT GCCTAATGAG TGAGCTAACT CACATTAATT GCGTTGCGCT CACTGCCCGC 
3241 TTTCCAGTCG GGAAACCTGT CGTGCCAGCT GCATTAATGA ATCGGCCAAC GCGCGGGGAG 
3301 AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC TGCGCTCGGT 
3361 CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG GTAATACGGT TATCCACAGA 
3421 ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG 
3481 TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG AGCATCACAA 
3541 AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT 
3601 TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT 
3661 GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAA TGCTCACGCT GTAGGTATCT 
3721 CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC CCGTTCAGCC 
3781 CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT 
3841 ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC 
3901 TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGGACAG TATTTGGTAT 
3961 CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT GATCCGGCAA 
4021 ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA 
4081 AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA 
4141 AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT 
4201 TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA 
4261 CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC 
4321 CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT TACCATCTGG 
4381 CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG GCTCCAGATT TATCAGCAAT 
4441 AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT CCGCCTCCAT 
4501 CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA ATAGTTTGCG 
4561 CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC TCGTCGTTTG GTATGGCTTC 
4621 ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA TCCCCCATGT TGTGCAAAAA 
4681 AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG CAGTGTTATC 
4741 ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT 
4801 TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC GGCGACCGAG 
4861 TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA CATAGCAGAA CTTTAAAAGT 
4921 GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA AGGATCTTAC CGCTGTTGAG 
4981 ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT TTACTTTCAC 
5041 CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC 
5101 GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA TATTATTGAA GCATTTATCA 
5161 GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA AACAAATAGG 
5221 GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC