pE2F-TA-Luc is designed to monitor the induction of E2F-mediated signal transduction pathways. E2F, a major target of the retinoblastoma gene (Rb), is a key regulator of cell-cycle checkpoints in mammalian cells (1–2). E2F plays a critical role in stimulating expression of genes encoding growth-promoting proteins (3), and is involved in regulating the expression of important genes during cell proliferation (4–5). The E2F protein forms a heterodimer complex with the DP1 protein, which binds to E2F response elements and initiates transcription of genes necessary for DNA replication. Studies have shown that deregulation of E2F results in a loss of cell-cycle checkpoints—thereby predisposing cells to uncontrolled growth (1–5). pE2F-TA-Luc contains four copies of the E2F enhancer element (6), located upstream of the minimal TA promoter, the TATA box from the herpes simplex virus thymidine kinase promoter (PTA). Located downstream of PTA is the firefly luciferase reporter gene (luc). Upon binding of the E2F/DP1 complex to the cis-acting E2F enhancer element, transcription is induced and the reporter gene is activated.

The luciferase coding sequence is followed by the SV40 late polyadenylation signal to ensure proper, efficient processing of the luciferase transcript in eukaryotic cells. A synthetic transcription blocker (TB) is located upstream of the cis-acting enhancer element. It is composed of adjacent polyadenylation and transcription pause sites for blocking nonspecific transcription (7). The vector backbone also contains an f1 origin for single-stranded DNA production, a pUC origin of replication, and an ampicillin resistance gene for propagation and selection in E. coli.

載體應用

pE2F-TA-Luc is designed for monitoring cell-cycle signaling in mammalian cells by assaying for luciferase activity. For example, induction of E2F-mediated signal transduction pathways may be compared across different cell types or cell states by transiently transfecting this vector into appropriate cell lines. After transfection, treat each culture individually with a drug candidate or stimulus of interest, then compare the activation of the E2F response element by assaying for the luciferase reporter gene. Additionally, you can monitor pathway activation by cotransfecting this vector with an expression vector containing a gene of interest. Luciferase is a highly sensitive enzymatic reporter that can be assayed by any standard luciferase-detection method, providing quantitative data on induction levels. pE2F-TA-Luc can be transfected into mammalian cells by any standard method. For selecting stable clones, cotransfect with a vector containing an antibiotic resistance gene, such as neomycin, hygromycin, or puromycin, and selecting resistant clones.

0001 GGTACCGAGC TCTTACGCGT GCTAGCCTTG GCGGGAGATA GAACTTGGCG GGAGATAGGA 
0061 ACTTGGCGGG AGATAGGAAC TTGGCGGGAG ATAGGAAAGA TCTGGGTATA TAATGGAAGC 
0121 TTGGCATTCC GGTACTGTTG GTAAAGCCAC CATGGAAGAC GCCAAAAACA TAAAGAAAGG 
0181 CCCGGCGCCA TTCTATCCGC TGGAAGATGG AACCGCTGGA GAGCAACTGC ATAAGGCTAT 
0241 GAAGAGATAC GCCCTGGTTC CTGGAACAAT TGCTTTTACA GATGCACATA TCGAGGTGGA 
0301 CATCACTTAC GCTGAGTACT TCGAAATGTC CGTTCGGTTG GCAGAAGCTA TGAAACGATA 
0361 TGGGCTGAAT ACAAATCACA GAATCGTCGT ATGCAGTGAA AACTCTCTTC AATTCTTTAT 
0421 GCCGGTGTTG GGCGCGTTAT TTATCGGAGT TGCAGTTGCG CCCGCGAACG ACATTTATAA 
0481 TGAACGTGAA TTGCTCAACA GTATGGGCAT TTCGCAGCCT ACCGTGGTGT TCGTTTCCAA 
0541 AAAGGGGTTG CAAAAAATTT TGAACGTGCA AAAAAAGCTC CCAATCATCC AAAAAATTAT 
0601 TATCATGGAT TCTAAAACGG ATTACCAGGG ATTTCAGTCG ATGTACACGT TCGTCACATC 
0661 TCATCTACCT CCCGGTTTTA ATGAATACGA TTTTGTGCCA GAGTCCTTCG ATAGGGACAA 
0721 GACAATTGCA CTGATCATGA ACTCCTCTGG ATCTACTGGT CTGCCTAAAG GTGTCGCTCT 
0781 GCCTCATAGA ACTGCCTGCG TGAGATTCTC GCATGCCAGA GATCCTATTT TTGGCAATCA 
0841 AATCATTCCG GATACTGCGA TTTTAAGTGT TGTTCCATTC CATCACGGTT TTGGAATGTT 
0901 TACTACACTC GGATATTTGA TATGTGGATT TCGAGTCGTC TTAATGTATA GATTTGAAGA 
0961 AGAGCTGTTT CTGAGGAGCC TTCAGGATTA CAAGATTCAA AGTGCGCTGC TGGTGCCAAC 
1021 CCTATTCTCC TTCTTCGCCA AAAGCACTCT GATTGACAAA TACGATTTAT CTAATTTACA 
1081 CGAAATTGCT TCTGGTGGCG CTCCCCTCTC TAAGGAAGTC GGGGAAGCGG TTGCCAAGAG 
1141 GTTCCATCTG CCAGGTATCA GGCAAGGATA TGGGCTCACT GAGACTACAT CAGCTATTCT 
1201 GATTACACCC GAGGGGGATG ATAAACCGGG CGCGGTCGGT AAAGTTGTTC CATTTTTTGA 
1261 AGCGAAGGTT GTGGATCTGG ATACCGGGAA AACGCTGGGC GTTAATCAAA GAGGCGAACT 
1321 GTGTGTGAGA GGTCCTATGA TTATGTCCGG TTATGTAAAC AATCCGGAAG CGACCAACGC 
1381 CTTGATTGAC AAGGATGGAT GGCTACATTC TGGAGACATA GCTTACTGGG ACGAAGACGA 
1441 ACACTTCTTC ATCGTTGACC GCCTGAAGTC TCTGATTAAG TACAAAGGCT ATCAGGTGGC 
1501 TCCCGCTGAA TTGGAATCCA TCTTGCTCCA ACACCCCAAC ATCTTCGACG CAGGTGTCGC 
1561 AGGTCTTCCC GACGATGACG CCGGTGAACT TCCCGCCGCC GTTGTTGTTT TGGAGCACGG 
1621 AAAGACGATG ACGGAAAAAG AGATCGTGGA TTACGTCGCC AGTCAAGTAA CAACCGCGAA 
1681 AAAGTTGCGC GGAGGAGTTG TGTTTGTGGA CGAAGTACCG AAAGGTCTTA CCGGAAAACT 
1741 CGACGCAAGA AAAATCAGAG AGATCCTCAT AAAGGCCAAG AAGGGCGGAA AGATCGCCGT 
1801 GTAATTCTAG AGTCGGGGCG GCCGGCCGCT TCGAGCAGAC ATGATAAGAT ACATTGATGA 
1861 GTTTGGACAA ACCACAACTA GAATGCAGTG AAAAAAATGC TTTATTTGTG AAATTTGTGA 
1921 TGCTATTGCT TTATTTGTAA CCATTATAAG CTGCAATAAA CAAGTTAACA ACAACAATTG 
1981 CATTCATTTT ATGTTTCAGG TTCAGGGGGA GGTGTGGGAG GTTTTTTAAA GCAAGTAAAA 
2041 CCTCTACAAA TGTGGTAAAA TCGATAAGGA TCCGTCGACC GATGCCCTTG AGAGCCTTCA 
2101 ACCCAGTCAG CTCCTTCCGG TGGGCGCGGG GCATGACTAT CGTCGCCGCA CTTATGACTG 
2161 TCTTCTTTAT CATGCAACTC GTAGGACAGG TGCCGGCAGC GCTCTTCCGC TTCCTCGCTC 
2221 ACTGACTCGC TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG 
2281 GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC 
2341 CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC 
2401 CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA 
2461 CTATAAAGAT ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC 
2521 CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT 
2581 AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG 
2641 CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC 
2701 AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA 
2761 GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT 
2821 AGAAGGACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT 
2881 GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG 
2941 CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG 
3001 TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA 
3061 AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA 
3121 TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG 
3181 ATCTGTCTAT TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA 
3241 CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG 
3301 GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT 
3361 GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT 
3421 TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC 
3481 TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA 
3541 TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT 
3601 AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC 
3661 ATGCCATCCG TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA 
3721 TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA 
3781 CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA 
3841 AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT 
3901 TCAGCATCTT TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC 
3961 GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA 
4021 TATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT 
4081 TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGCG 
4141 CCCTGTAGCG GCGCATTAAG CGCGGCGGGT GTGGTGGTTA CGCGCAGCGT GACCGCTACA 
4201 CTTGCCAGCG CCCTAGCGCC CGCTCCTTTC GCTTTCTTCC CTTCCTTTCT CGCCACGTTC 
4261 GCCGGCTTTC CCCGTCAAGC TCTAAATCGG GGGCTCCCTT TAGGGTTCCG ATTTAGTGCT 
4321 TTACGGCACC TCGACCCCAA AAAACTTGAT TAGGGTGATG GTTCACGTAG TGGGCCATCG 
4381 CCCTGATAGA CGGTTTTTCG CCCTTTGACG TTGGAGTCCA CGTTCTTTAA TAGTGGACTC 
4441 TTGTTCCAAA CTGGAACAAC ACTCAACCCT ATCTCGGTCT ATTCTTTTGA TTTATAAGGG 
4501 ATTTTGCCGA TTTCGGCCTA TTGGTTAAAA AATGAGCTGA TTTAACAAAA ATTTAACGCG 
4561 AATTTTAACA AAATATTAAC GTTTACAATT TCCCATTCGC CATTCAGGCT GCGCAACTGT 
4621 TGGGAAGGGC GATCGGTGCG GGCCTCTTCG CTATTACGCC AGCCCAAGCT ACCATGATAA 
4681 GTAAGTAATA TTAAGGTACG GGAGGTACTT GGAGCGGCCG CAATAAAATA TCTTTATTTT 
4741 CATTACATCT GTGTGTTGGT TTTTTGTGTG AATCGATAGT ACTAACATAC GCTCTCCATC 
4801 AAAACAAAAC GAAACAAAAC AAACTAGCAA AATAGGCTGT CCCCAGTGCA AGTGCAGGTG 
4861 CCAGAACATT TCTCTATCGA TA