title: Application of NanoLuc to monitor the intrinsic promoter activity of GRP78 using the CRISPR/Cas9 system.
abstract: In this study, we applied a highly sensitive small luciferase, NanoLuc, to establish a knock-in cell line using the CRISPR/Cas9 system and characterized the endogenous promoter activity of the glucose-regulated protein 78 (GRP78) gene. The N-terminal region of the human GRP78 gene was fused to the NanoLuc gene and aligned with the puromycin-resistant gene through the 2A peptide sequence and used as a knock-in vector. The selected cells responded to both pharmacological and genetic ER stress and show NanoLuc-based CRISPR/Cas9 system is a very useful tool to isolate gene-edited cells and to characterize the endogenous promoter activity for genes of interest.
authors: United Graduate School of Drug Discovery and Medical Information Science, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. oohashi@gifu-u.ac.jp.|Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. oohashi@gifu-u.ac.jp.
authors: Kentaro
authors: K
authors: Oh-Hashi
authors: Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors: Eri
authors: E
authors: Furuta
authors: United Graduate School of Drug Discovery and Medical Information Science, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors: Junpei
authors: J
authors: Norisada
authors: Department of Anesthesiology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, 602-0841, Japan.
authors: Fumimasa
authors: F
authors: Amaya
authors: United Graduate School of Drug Discovery and Medical Information Science, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.|Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors: Yoko
authors: Y
authors: Hirata
authors: United Graduate School of Drug Discovery and Medical Information Science, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.|Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors: Kazutoshi
authors: K
authors: Kiuchi
mesh_terms: D051702:Activating Transcription Factor 6 / Q000235:genetics; D000818:Animals; D064113:CRISPR-Cas Systems; D002460:Cell Line; D000091342:Endoplasmic Reticulum Chaperone BiP; D055879:Gene Knock-In Techniques; D057809:HEK293 Cells; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D049408:Luminescent Agents; D051379:Mice; D011401:Promoter Regions, Genetic; D000094704:RNA, Guide, CRISPR-Cas Systems; D011993:Recombinant Fusion Proteins / Q000235:genetics
chemical_list: D051702:Activating Transcription Factor 6; C497112:Atf6 protein, mouse; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D049408:Luminescent Agents; D000094704:RNA, Guide, CRISPR-Cas Systems; D011993:Recombinant Fusion Proteins