Abstract
We introduce a simple and universal cloning plasmid system for gene expression in prokaryotic (Escherichia coli) and mammalian cells. This novel system has two expression modes: the (subcloning) prokaryotic and mammalian modes. This system streamlines the process of producing mammalian gene expression plasmids with desired genes. The plasmid (prokaryotic mode) has an efficient selection system for DNA insertion, multiple component genes with rare restriction sites at both ends (termed “units”), and a simple transformation to mammalian expression mode utilizing rare restriction enzymes and re-ligation (deletion step). The new plasmid contains the lac promoter and operator followed by a blunt-end EcoRV recognition site, and a DNA topoisomerase II toxin-originated gene for effective selection with isopropyl-β-D-thiogalactoside (IPTG) induction. This system is highly efficient for the subcloning of blunt-end fragments, including PCR products. After the insertion of the desired gene, protein encoded by the desired gene can be detected in E. coli with IPTG induction. Then, the lac promoter and operator are readily deleted with 8-nucleotide rare-cutter blunt-end enzymes (deletion step). Following re-ligation and transformation, the plasmid is ready for mammalian expression analysis (mammalian mode). This idea (conversion from prokaryotic to mammalian mode) can be widely adapted. The pgMAX system overwhelmingly simplifies prokaryotic and mammalian gene expression analyses.
Highlights
Numerous commercial expression plasmids exist, especially for mammalian transient expression
The pgMAX plasmid was based on pIRESpuro3 (Clontech)
The pgMAX plasmid has two functional components, the prokaryotic component for prokaryotic gene expression and for efficient subcloning (Fig 1 prokaryotic unit) and the mammalian expression component, which is composed of the CMV promoter and the internal ribosomal entry site (IRES) puromycin N-acetyl-transferase (Pac) with a polyA tail
Summary
Numerous commercial expression plasmids exist, especially for mammalian transient expression. A simple subcloning and expression plasmid of a polymerase chain reaction (PCR) product with T4 DNA polymerase, ligating a fragment into a blunt-end EcoRV site in a pBluescript plasmid, or restriction enzyme digestion and ligation. TA cloning uses Taq DNA polymerase to add a single adenosine-residue overhang to the 3’ end of the PCR product. For rapid construction of multiple DNA fragment ligations, both Gibson Assembly and Golden Gate Assembly are available, they require specific enzymes [6, 7]. Another ligation method for multiple DNA fragments, the SLIC method, has been established [8] This method is cost effective and dependent on PCR and T4 exonuclease activity
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