A simple and dual expression plasmid system in prokaryotic (E. coli) and mammalian cells

• Published in: PloS one Vol. 14; no. 5; p. e0216169
• Main Authors: Murakami, Manabu, Ohba, Takayoshi, Murakami, Agnieszka M, Han, Chong, Kuwasako, Kenji, Itagaki, Shirou
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 02.05.2019; Public Library of Science (PLoS)
• Subjects: Adenosine; Animal genetics; Animals; Base Sequence - genetics; Biochemistry; Biology and life sciences; Cell culture; Cells (Biology); Clonal deletion; Cloning; Cloning, Molecular - methods; Deoxyribonucleic acid; DNA; DNA polymerase; DNA Restriction Enzymes - genetics; DNA topoisomerase; DNA topoisomerase (ATP-hydrolysing); E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli Proteins - genetics; Gene deletion; Gene expression; Gene Expression - genetics; Genes; Genetic aspects; Genetic research; Genetic transformation; Genetic Vectors - genetics; Insertion; Isopropyl thiogalactoside; Mammalian cells; Mammals; Mammals - genetics; Mammals - metabolism; Medicine; Methods; Neomycin; Novels; Pharmacology; Physical Sciences; Plasmids; Plasmids - genetics; Prokaryotes; Prokaryotic Cells - metabolism; Promoter Regions, Genetic - genetics; Protein Engineering - methods; Research and analysis methods; Streamlines; Topoisomerases; Transformation; University graduates; United States > US; Japan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0216169

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.