A simple and dual expression plasmid system in prokaryotic (E. coli) and mammalian cells
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2019 |
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| Erschienen: |
2019 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:14 |
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| Enthalten in: |
PloS one - 14(2019), 5 vom: 03., Seite e0216169 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Murakami, Manabu [VerfasserIn] |
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| Links: |
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| Themen: |
DNA Restriction Enzymes |
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| Anmerkungen: |
Date Completed 20.02.2020 Date Revised 09.03.2020 published: Electronic-eCollection Citation Status MEDLINE |
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| doi: |
10.1371/journal.pone.0216169 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM296684341 |
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| 245 | 1 | 2 | |a A simple and dual expression plasmid system in prokaryotic (E. coli) and mammalian cells |
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| 500 | |a Date Revised 09.03.2020 | ||
| 500 | |a published: Electronic-eCollection | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
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| 650 | 7 | |a DNA Restriction Enzymes |2 NLM | |
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| 700 | 1 | |a Ohba, Takayoshi |e verfasserin |4 aut | |
| 700 | 1 | |a Murakami, Agnieszka M |e verfasserin |4 aut | |
| 700 | 1 | |a Han, Chong |e verfasserin |4 aut | |
| 700 | 1 | |a Kuwasako, Kenji |e verfasserin |4 aut | |
| 700 | 1 | |a Itagaki, Shirou |e verfasserin |4 aut | |
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