p53 The p53 Dominant-Negative Vector Set lets you study p53 and related signaling pathways. These vectors constitutively express high levels of wild-type p53 or its dominant-negative mutant. To determine the effects of p53 expression on a particular cell line, simply transfect with the vector containing the gene for wild-type p53, and examine morphology or look for changes in the expression of other pathway proteins. 载体描述 The p53 Dominant-Negative Vector Set is a convenient tool for monitoring p53-mediated signal transduction pathways. This vector set consists of two vectors, pCMV-p53 and pCMV-p53mt135. pCMV-p53 expresses the wild-type p53 tumor suppression protein and pCMV-p53mt135 expresses a dominant-negative mutant. Both proteins are expressed at high levels from the constitutive CMV promoter. The p53 gene and p53mt135 gene differ by a G to A conversion at nucleotide 1017.
The SV40 polyadenylation sequence directs proper processing of the 3' end of the p53 or p53mt135 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) gene—allows 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 pCMV-p53 is used to examine the physical interactions between p53 and other proteins, to induce p53-mediated cell cycle arrest, or to study p53-induced gene expression. pCMV-p53mt135 expresses the p53mt135 mutant, which because of a conformational change, can no longer interact with p53-binding sites. When p53mt135 and p53 are co-expressed, they form a mixed tetramer that is unable to interact with p53-binding sites; therefore, the downstream effects of p53 are blocked (1, 2).
Both 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: DH5a 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 (50μg/ml) in E. coli hosts.
E. coli replication origin: pUC
Copy number: ~500
Plasmid incompatibility group: pMB1/ColE1
