Department of Pharmacology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
Department of Cell Physiology, Akita University Graduate School of Medicine, Akita, Japan.
PLoS One. 2019 May 2;14(5):e0216169. doi: 10.1371/journal.pone.0216169. eCollection 2019.
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.
我们介绍了一种用于原核（大肠杆菌）和哺乳动物细胞中基因表达的简单而通用的克隆质粒系统。该新型系统具有两种表达模式：（亚克隆）原核模式和哺乳动物模式。该系统简化了具有所需基因的哺乳动物基因表达质粒的生产过程。该质粒（原核模式）具有高效的 DNA 插入选择系统、两端稀有限制酶切位点的多组分基因（称为“单元”），以及利用稀有限制酶和再连接（缺失步骤）转化为哺乳动物表达模式的简单方法。新质粒包含 lac 启动子和操纵子，随后是平末端 EcoRV 识别位点，以及 DNA 拓扑异构酶 II 毒素起源基因，用于异丙基-β-D-硫代半乳糖苷（IPTG）诱导的有效选择。该系统对于平末端片段（包括 PCR 产物）的亚克隆非常高效。在插入所需基因后，可通过 IPTG 诱导在大肠杆菌中检测到所需基因编码的蛋白质。然后，用 8 个核苷酸的稀有切割平末端酶（缺失步骤）很容易删除 lac 启动子和操纵子。重新连接和转化后，该质粒即可用于哺乳动物表达分析（哺乳动物模式）。这种思路（从原核模式转换为哺乳动物模式）可以广泛适应。pgMAX 系统极大地简化了原核和哺乳动物基因表达分析。
