pcDNA Plasmids
pcDNA Plasmids are a family of mammalian expression vectors widely used in molecular biology for the transient or stable expression of recombinant proteins in mammalian cells. Developed by Invitrogen (now part of Thermo Fisher Scientific), these plasmids are designed to provide high levels of protein expression driven by the strong human cytomegalovirus (CMV) immediate-early promoter.
pcDNA Plasmids: Technical Insights
- Structure and Components:
- Origin of Replication (ori): pcDNA plasmids contain the ColE1 origin of replication for propagation in Escherichia coli, enabling high plasmid yields for transfection experiments.
- Antibiotic Resistance Gene:
- ampR: Provides resistance to ampicillin, allowing for the selection of bacteria that have taken up the plasmid.
- CMV Promoter: The human CMV immediate-early promoter drives high-level expression of the gene of interest in mammalian cells. This promoter is strong and constitutive, ensuring robust expression across various cell types.
- Multiple Cloning Site (MCS): The MCS contains several unique restriction enzyme sites, facilitating the insertion of the gene of interest. The site is positioned downstream of the CMV promoter for efficient transcription.
- Polyadenylation Signal (polyA): A synthetic bovine growth hormone (BGH) polyadenylation signal downstream of the MCS ensures proper mRNA processing and stability.
- SV40 Origin: The SV40 origin of replication is included to allow for episomal replication in cells expressing the SV40 large T antigen, such as COS-7 cells, enhancing plasmid copy number and expression.
- Neomycin Resistance Gene (neoR): Provides resistance to G418 (geneticin) in mammalian cells, allowing for the selection of stably transfected cells.
- Applications:
- Protein Expression: pcDNA plasmids are primarily used for the expression of recombinant proteins in mammalian cells. The strong CMV promoter ensures high levels of expression, suitable for functional studies, protein-protein interaction assays, and biochemical analyses.
- Gene Function Studies: By expressing wild-type or mutant genes, researchers can study the effects on cellular pathways, identify gene
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