干旱区生态环境知识资源中心(Arid): Development of a Tet-On Inducible Expression System for the Anhydrobiotic Cell Line, Pv11

Arid

DOI	10.3390/insects11110781
	Development of a Tet-On Inducible Expression System for the Anhydrobiotic Cell Line, Pv11
	Tokumoto, Shoko; Miyata, Yugo; Usui, Kengo; Deviatiiarov, Ruslan; Ohkawa, Takahiro; Kondratieva, Sabina; Shagimardanova, Elena; Gusev, Oleg; Cornette, Richard; Itoh, Masayoshi; Hayashizaki, Yoshihide; Kikawada, Takahiro
通讯作者	Kikawada, T
来源期刊	INSECTS
EISSN	2075-4450
出版年	2020
卷号	11 期号:11
英文摘要	Simple Summary Anhydrobiosis is a unique ametabolic state that is characterized by extreme body water loss due to desiccation, generally over 95%. The sleeping chironomid, Polypedilum vanderplanki, is an anhydrobiotic insect inhabiting semi-arid regions in Africa. Pv11 is an anhydrobiotic insect cell line established from the P. vanderplanki larvae, and the cell line also displays the extreme desiccation tolerance. This unique feature could be useful for developing a dry preservation technique. However, effective expression systems for protein production have not been available due to a limited number of experimental tools in Pv11 cells. To solve this problem, we identified a minimal promoter from the genome sequence of P. vanderplanki because promoters designed for model organisms are not available in Pv11 cells, although minimal promoters are commonly used for molecular biology experiments and biotechnology fields. The identification of the minimal promoter allowed us to develop an effectively inducible expression system in Pv11 cells. By using the system, several proteins including a functional protein with complex structures were successfully expressed in the cells. The successful establishment of the inducible system on Pv11 cells could contribute to bio-industrial protein production. The Pv11 cell line established from an African chironomid, Polypedilum vanderplanki, is the only cell line tolerant to complete desiccation. In Pv11 cells, a constitutive expression system for Pv11 cells was previously exploited and several reporter genes were successfully expressed. Here we report the identification of an effective minimal promoter for Pv11 cells and its application to the Tet-On inducible expression system. First, using a luciferase reporter assay, we showed that a 202 bp deletion fragment derived from the constitutively active 121-promoter functions in Pv11 cells as an appropriate minimal promoter with the Tet-On inducible expression system. The AcGFP1 (Aequorea coerulescens green fluorescent protein) was also successfully expressed in Pv11 cells using the inducible system. In addition to these reporter genes, the avian myeloblastosis virus reverse transcriptase alpha subunit (AMV RT alpha), which is one of the most widely commercially available RNA-dependent DNA polymerases, was successfully expressed through the inducible expression system and its catalytic activity was verified. These results demonstrate the establishment of an inducible expression system in cells that can be preserved in the dry state and highlight a possible application to the production of large and complex proteins.
英文关键词	anhydrobiosis desiccation tolerance Polypedilum vanderplanki insect cells minimal promoter Tet-On system
类型	Article
语种	英语
开放获取类型	Green Published, Green Submitted, gold
收录类别	SCI-E
WOS记录号	WOS:000593374300001
WOS关键词	PROTEIN MODEL PEPTIDES ; RNA INTERFERENCE ; CORE PROMOTER ; GENE ; INSECT ; POLYMERASE ; ESTABLISHMENT ; VACCINES ; PLATFORM
WOS类目	Entomology
WOS研究方向	Entomology
资源类型	期刊论文
条目标识符	http://119.78.100.177/qdio/handle/2XILL650/327704
作者单位	[Tokumoto, Shoko; Cornette, Richard; Kikawada, Takahiro] Univ Tokyo, Grad Sch Frontier Sci, Kashiwa, Chiba 2778562, Japan; [Miyata, Yugo; Kikawada, Takahiro] Natl Inst Agr & Food Res Org NARO, Inst Agrobiol Sci, Tsukuba, Ibaraki 3050851, Japan; [Usui, Kengo; Ohkawa, Takahiro] RIKEN, Ctr Integrat Med Sci IMS, Yokohama, Kanagawa 2300045, Japan; [Deviatiiarov, Ruslan; Kondratieva, Sabina; Shagimardanova, Elena; Gusev, Oleg] Kazan Fed Univ, Inst Fundamental Med & Biol, Kazan TATARSTAN, Russia; [Gusev, Oleg; Itoh, Masayoshi; Hayashizaki, Yoshihide] RIKEN, Prevent Med & Diag Innovat Program, Wako, Saitama 3510198, Japan
推荐引用方式 GB/T 7714	Tokumoto, Shoko,Miyata, Yugo,Usui, Kengo,et al. Development of a Tet-On Inducible Expression System for the Anhydrobiotic Cell Line, Pv11[J],2020,11(11).
APA	Tokumoto, Shoko.,Miyata, Yugo.,Usui, Kengo.,Deviatiiarov, Ruslan.,Ohkawa, Takahiro.,...&Kikawada, Takahiro.(2020).Development of a Tet-On Inducible Expression System for the Anhydrobiotic Cell Line, Pv11.INSECTS,11(11).
MLA	Tokumoto, Shoko,et al."Development of a Tet-On Inducible Expression System for the Anhydrobiotic Cell Line, Pv11".INSECTS 11.11(2020).