載體描述
CREB (CRE-binding protein) is a member of the leucine zipper family of transcription factors and forms both a homodimer with itself and heterodimers with other leucine zipper proteins (1, 2). CREB also has a kinase-inducible domain, which contains consensus phosphorylation sites for several kinases, such as protein kinase A (1, 2). The CREB Dominant-Negative Vector Set consists of three vectors:
pCMV-CREB Vector onstitutively expresses the human wild-type (wt) CREB protein. pCMV-CREB133 Vector expresses a mutant variant of the human CREB protein that contains a serine to alanine mutation corresponding to amino acid 133 in the mutant mouse CREB protein. This mutation blocks phosphorylation of CREB, thus preventing transcription.
pCMV-KCREB Vector expresses a mutant variant of the human CREB protein that contains mutations in its DNA-binding domain. KCREB acts as a dominant repressor by forming an inactive dimer with CREB, blocking its ability to bind cAMP-regulated enhancer element (CRE).
These proteins are expressed at high levels from the constitutive CMV promoter. The SV40 polyadenylation sequence directs proper processing of the 3' end of the mRNAs. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neor) consisting of the SV40 early promoter, the Tn5 neomycin/kanamycin resistance gene, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gen eallows kanamycin selection in E. coli and neomycin
selection in eukaryotic cells. The vector backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
使用
The CREB Dominant-Negative Vector Set can be used to monitor signal transduction pathways related to CREB. In conjunction with reporter systems (such as our cis-acting reporter vectors), you can monitor CREB-induced transcription by assaying for the reporter. For example, CREB activation can be measured in cells cotransfected with pCMV-CREB and pCRE-d2EGFP (Cat.No. 631802) by treating the transfected cells with forskolin and observing EGFP expression by FACS analysis or fluorescence microscopy. Cells expressing dominant-negative CREB133 or KCREB will not respond to this stimulus, so induced transcription by forskolin is inhibited. For more information about Pathway Profiling Vectors, visit our web site at www.clontech.com.
These vectors can be transfected into mammalian cells using any standard method. Stable transformants can be selected using G418(3).
Propagation in E. coli
Suitable host strains: DH5α, HB101 and other general purpose strains. Single-stranded DNA production requires a host containing an F plasmid such as JM101 or XL1-Blue.
Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) to E. coli hosts.
E. coli replication origin: pUC
Copy number: ~500
Plasmid incompatibility group: pMB1/ColE1
