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复制子 | pUC |
原核抗性 | Amp |
真核抗性 | 荧光素酶Luc |
克隆菌株 | DH5α |
培养条件 | 37℃ |
质粒宿主 | 哺乳细胞 |
质粒用途 | 信号报告 |
原核抗性 | 氨苄青霉素 |
荧光标记 | Fluc |
pE2F-TA-Luc单荧光素酶信号通路报告质粒。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.
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