Introduction on pcDNA3.1(+)/pcDNA3.1 (-) Vectors

In the field of pharmaceuticals and biotechnology studies, we generally use the plasmid vector to deliver the target genes into mammalian cells by transfection method in the laboratory. Based on the functions and usages, plasmid vectors are usually divided into cloning vectors and expression vectors. Cloning vectors are mainly used to clone and amplify DNA fragments, so that foreign genes can be replicated and amplified in host cells. Expression vectors are used to efficiently express foreign genes in host cells. In addition to the function of replication and amplification just like cloning vectors, the expression vectors usually contain DNA elements necessary for transcription and translation. Here we would like to introduce pcDNA3.1(+)/pcDNA3.1 (-)vector commonly used in mammalian cells.

pcDNA3.1(+) and pcDNA3.1(-) are 5.4 kb vectors derived from pcDNA3 and designed for high-level stable and transient expression in mammalian hosts. The high-level stable and non-replicative transient expression can be carried out in most mammalian cells. Therefore, these two vectors play an important role in the study of protein structure and function research. 

pcDNA3.1(+) and pcDNA3.1(-)vectors contain the following elements:

l  CMV promoter: Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a wide range of mammalian cells. The CMV promoter fragment in the vector is only over 0.5 kb. It has a powerful starting function and is usually used to express proteins or commercial recombinant antibodies in a variety of mammalian cells (such as CHO cells, HEK293 cells, BHK cells, etc.).

l  MCS: The multi-cloning site (MCS) area contains 10 commonly used restriction sites such as BamHI, EcoRI, etc. The selection of various sites can facilitate the insertion of foreign DNA fragments and easy to manipulate gene cloning.

l  BGH pA/SV40 pA: BGH pA/SV40 pA can terminate translation expression. These 2 elements contain the AAUAAA motif to promote polyadenylation and transcription termination, making the target gene more stable after transcription.

l  Neo: Neomycin resistance gene, it is started by SV40 early promoter. After transfection into cells, drug screening is performed in the culture medium to kill cells that do not have neomycin resistance and retain cells that already harbored the plasmids.

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

l  Amp: Ampicillin resistance gene which allows the plasmid to maintain by ampicillin selection in E.coli.

Application of pcDNA3.1 vectors

An article published in the Nature microbiology reported an approach to rapidly screen lineage B betacoronaviruses, such as SARS-CoV and the recent SARS-CoV-2, for receptor usage and their ability to infect cell types from different species. The research shows that host protease processing during viral entry is a significant barrier for several lineage B viruses and that bypassing this barrier allows several lineage B viruses to enter human cells through an unknown receptor. In addition, the research demonstrates how different lineage B viruses can recombine to gain entry into human cells, and confirm that human ACE2 is the receptor for the recently emerging SARS-CoV-2. GenScript helps the customer synthesize the ACE2 gene and clone it into the pcDNA3.1+ vector.

Reference: Michael Letko et al., (2020) Functionalassessment of cell entry and receptor usage for SARS-CoV-2 and other lineage Bbetacoronaviruses. Nature Microbiology. DOI: 10.1038/s41564-020-0688-y