Mammalian Gene Expression pcDNA3.1 Vector

Overview

pcDNA3.1 is a widely used mammalian expression vector designed for plasmid transfection and robust, constitutive expression of genes of interest in mammalian cells. While many conventional plasmids are designed to enable bacterial cloning, the pcDNA3.1 expression vector has been specialized for transfection into mammalian cells for transgene expression as well as for production of ssDNA for incorporation into phage particles and production of in vitro transcribed (IVT) mRNA.

A comparison of the major components found in pRP (VectorBuilder’s regular plasmid backbone) and pcDNA3.1 vectors is provided in the table below.

For further information about this vector system, please refer to the papers below.

Highlights

VectorBuilder’s pcDNA3.1 vector has multiple distinguishing features to facilitate efficient transfection and expression in mammalian cells. Notably, a T7 promoter enables in vitro transcription of the downstream ORF; additionally, a phage-derived f1 origin of replication allows replication and packaging of ssDNA into phage particles.

Advantages

Technical simplicity: Mammalian cell transfection of plasmids  is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.

High-level expression: pcDNA3.1 transfection can often result in very high copy numbers in cells. This can lead to very high, constitutive expression of transgenes carried on the vector.

Compatible with various applications: Inclusion of the phage-derived f1 origin of replication and the T7 promoter allow for production and packaging of ssDNA into phage particles and in vitro transcription of mRNA, respectively.

Disadvantages

Non-integration of vector DNA: Conventional transfection of plasmid vectors is also referred to as transient transfection because the vast majority of vector exists as episomal DNA. However, very infrequently (one per 10² to 10⁶ cells depending on cell type), plasmid DNA can integrate permanently into the host genome. If a drug resistance or fluorescence marker is incorporated into the plasmid, cells stably integrating the plasmid can be derived by drug selection or cell sorting after extended culture.

Limited cell type range: The efficiency of plasmid transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. This is compounded by the fact that the CMV promoter is potentially vulnerable to methylation-induced transgene silencing in a cell type-specific manner. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo.

Non-uniformity of gene delivery: Although a successful transfection can result in very high copy number of the transfected plasmid vector per cell, this can be highly non-uniform. Some cells can carry many copies while others carry very few or none. This is unlike transduction by virus-based vectors which tends to result in relatively uniform gene delivery into cells.

Key components

CMV Promoter: The human cytomegalovirus (CMV) promoter is placed here.

T7 Promoter: Enables in vitro transcription of the incorporated gene of interest.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

BGH late pA: Triggers transcription termination and increases stability of transcribed mRNA.

f1 ori: Allows replication and packaging of ssDNA into phage particles.

SV40 promoter: Simian virus 40 promoter. It drives expression of the downstream marker gene.

Neomycin: Neomycin resistance gene. It allows cells to be resistant to geneticin (G418).

SV40 pA: SV40 polyadenylation signal. It facilitates transcriptional termination of the upstream marker gene.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

Representative vector design