pCMV-MEKK1載體使用說(shuō)明: pCMV-MEKK1 is designed for the constitutive expression of MEK Kinase 1 (MEKK1) in mammalian cells. It can also be used to study the effects of a given stimulus on the JNK/SAPK pathway. In conjunction with Clontech’s In Vivo Kinase Assay Kits, pCMV-MEKK1 is an ideal positive control for studying a gene or molecule and its affect on kinase activation in the JNK/SAPK pathway.
MEKK1 mediates cellular responses to diverse stimuli, such as cytokines, growth factors, and other stressors via activation of JNK/SAPK signaling pathway. It has also been reported that MEKK1 is a possible regulator in signal transduction pathways involving apoptosis (1). To a lesser extent, MEKK1 can also activate the ERK pathway via phosphorylation of MEK (2, 3); however, it is more efficient in activating the JNK/SAPK than ERK (4, 5).
pCMV-MEKK1 contains a gene encoding MEKK1 (mouse) that is driven by the human cytomegalovirus(CMV) promoter. SV40 polyadenylation signals downstream of the MEKK1 gene direct proper processing of the 3’ end of the mRNA. The vector backbone also 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 neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene—allows stably transfected eukaryotic cells to be selected using G418(6). A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pCMV-MEKK1 backbone also provides a pUC origin of replication for propagation in E.coli and an f1 origin for single-stranded DNA production. The recombinant vector can be transfected into mammalian cells using any standard method.
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 JM109 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
