■ 基本信息

■ 質粒屬性

■ 質粒簡介

pE2F-TA-Luc單熒光素酶信號通路報告質粒。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 . E2F plays a critical role in stimulating expression of genes encoding growth-promoting proteins, and is involved in regulating the expression of important genes during cell proliferation . 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 . 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.

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.

■ 質粒圖譜

■ 質粒序列

質粒序列請下載：

測序得的部分序列

       1 acatttctct atcgataggt accgagctct tacgcgtgct agccttggcg ggagatagaa

      61 cttggcggga gataggaact tggcgggaga taggaacttg gcgggagata ggaaagatct

     121 agactctaga gggtatataa tggaagctcg aattccagct tggcattccg gtactgttgg

     181 taaaaagctt ggcattccgg tactgttggt aaagccacca tggaagacgc caaaaacata

     241 aagaaaggcc cggcgccatt ctatccgctg gaagatggaa ccgctggaga gcaactgcat

     301 aaggctatga agagatacgc cctggttcct ggaacaattg cttttacaga tgcacatatc

     361 gaggtggaca tcacttacgc tgagtacttc gaaatgtccg ttcggttggc agaagctatg

     421 aaacgatatg ggctgaatac aaatcacaga atcgtcgtat gcagtgaaaa ctctcttcaa

     481 ttctttatgc cggtgttggg cgcgttattt atcggagttg cagttgcgcc cgcgaacgac

     541 atttataatg aacgtgaatt gctcaacagt atgggcattt cgcagcctac cgtggtgttc

     601 gtttccaaaa aggggttgca aaaaattttg aacgtgcaaa aaaagctccc aatcatccaa

     661 aaaattatta tcatggattc taaaacggat taccagggat ttcagtcgat gtacacgttc

     721 gtcacatctc atctacctcc cggttttaat gaatacgatt ttgtgccaga gtccttcgat

     781 agggacaaga caattgcact gatcatgaac tcctctggat ctactggtct gcctaaaggt

     841 gtcgctctgc ctcatagaac tgcctgcgtg agattctcgc atgccagaga tcctattttt

     901 ggcaatcaaa tcattccgga tactgcgatt ttaagtgttg ttccattcca tcacggtttt

     961 ggaatgttta ctacactcgg atatttgata tgtggatttc gagtcgtctt aatgtataga

    1021 tttgaagaag agctgtttct gaggagcctt cagattacag gattcaaagt gcgctgctgg

    1081 tgcaacccta attctcttct cgcaaagcac tctgatgaca atcgagttta tctaaattta

    1141 acacgaatgg cttctgggtg ccgctcccct ctttaggagt cgggaggcga ttgccaagag

    1201 tcaatctgcc aggcatcagg gcaagagaat attgggcatc gctgtcgaag gacatacact