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PHOTOACTIVATABLE TET EXPRESSION CONTROL SYSTEM NEW

外国特許コード F200010259
整理番号 K108P26WO
掲載日 2020年11月9日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2019JP034217
国際公開番号 WO2020045651
国際出願日 令和元年8月30日(2019.8.30)
国際公開日 令和2年3月5日(2020.3.5)
優先権データ
  • 特願2018-163617 (2018.8.31) JP
発明の名称 (英語) PHOTOACTIVATABLE TET EXPRESSION CONTROL SYSTEM NEW
発明の概要(英語) The present invention provides a photoactivatable Tet-Off/On system enabling accurate temporal and spatial control of gene expression. The present invention is a PA-Tet-Off/On system comprising: a target gene expression cassette provided with a TRE that contains a TetO sequence, a promoter that is controlled by the TRE, and a target gene wherein the expression of said gene is controlled by the promoter; a first fusion protein expression cassette provided with a gene that codes for a first fusion protein containing TetR or rTetR and a first protein; and a second fusion protein expression cassette provided with a gene that codes for a second fusion protein containing p65AD and a second protein, wherein the first protein and the second protein bind together to form a heterodimer only when irradiated at a specific wavelength.
従来技術、競合技術の概要(英語) BACKGROUND ART
Tet-OFF/ON system, Tet operator sequence (TetO) Tet repressor (TRE) and Tet response factors (TetR) are used for the interaction, a more stable and Tet (Dox) doxycycline Tet analog by processing, in a target cell to modulate the expression of the exogenous gene (for example, see Non-Patent Document 1.). Tet-OFF system, in the absence of Dox, TetR fusion protein and the transcriptional activation domain is coupled to the TRE, downstream of the minimum gene expression promoter is activated. Tet-ON system, in the presence of a Dox, reverse transcriptional activation domain and TetR (rTetR) coupled to the TRE of the fusion protein, a minimum downstream gene expression promoter is activated. Tet-OFF/ON system, the most commonly used in mammalian cells chemically in a controlled system, a small molecule expression regulatory Dox for this reason, the expression of target genes, for a limited time frame and, only in cells within a limited space is difficult to perform. For example, an individual occurrence and maintenance of stem cells maintain tissue homeostasis, proliferation, cell differentiation in the case of the, stem or progenitor cell gene expression plays an important role in dynamic role has been known. In addition, these phenomena, the circadian rhythm or clock is about the function of the gene-rhythms is a close correlation is known. However, the Tet-OFF/ON system, these studies required adaptation of the temporal resolution, spatial resolution may be impossible to perform an operation, such as the rapid activation of a gene of interest or need to deactivate the research, is not suitable for the experiment.
In a conventional gene expression is controlled to overcome the technical limitations of the system, temporal, spatial control can be achieved as a system for gene expression, gene expression (ON/OFF) by light irradiation can be controlled, that is photoactivatable (photoactivatable,PA) expression system has been developed. By performing the control of gene expression with light, the light irradiation area as well as adjusting the radiation intensity, only the cells in a particular space, in a limited time frame, the expression of target genes can be easily derived. For example, in the light activated transcription factor PA-Gal4/UAS GAVPO (Light-ON system) using the system, basic helix loop, helix (bHLH) transfer of the functional importance of the dynamic changes in gene expression analysis has been reported (Non-Patent Document 2 and Non-Patent Document 3). GAVPO is activated and deactivated for a rapid reaction rate, by changing the light irradiation pattern, neural stem cell gene expression in various kinetics Ascl1 (for example, to continuously or vibration) can be directed to a man.
Proteins that interact with the light-dependent module, a blue-responsive from the Arabidopsis (Arabidopsis thaliana) hetero-dimer formation and module. The module comprises a light receptor and its specific binding proteins, Cry2(Cryptochrome 2) is made CIB1(cryptochrome-interacting basic helix-loop-helix 1) (for example, Non-Patent Document 4-8). Arabidopsis thaliana Cry2 is, via regulation of the circadian rhythm to modulate growth and development of the plant the photolyase-like light receptor. Cry2 is, N photopolyase homology region (PHR) and the end of the terminal extension 2 of the (CCE or CCT) Cryptochrome C of the two domains. The PHR, flavin adenine dinucleotide (FAD) chromophore in the chromophore-binding domain of non-covalent. Cry2 is, specific bHLH transcription factor to the blue light CIB1 can be coupled. Cry2 and essential domain truncated is CIB1, the hetero-dimer formation on the blue light which acts as a module. In addition, some point mutations Cry2, faster or slower the light cycle was shown to induce (Non-Patent Document 9, Non-Patent Document 10, Non-Patent Document 11).
In addition, the near-infrared photoresponsive hetero-dimer formation as a module, and the photosynthesis bacteria rod pseudomonas, phytochrome BphP1 (Rhodopseudomonaspalustris) pulse is derived from tris-specific binding protein and a protein PpsR2 and modules (for example, Non-Patent Document 27). 740-780nm PpsR2 BphP1 and to the near-infrared light is irradiated, both may be bonded to form a heterodimer. The heterodimer, including eukaryotic and mammalian endogenous chromophore Biliverdin (BV) may be used to absorb the near-infrared light is formed. In addition, a large number of domain PpsR2 relatively large protein. Therefore, to improve the BphP1/PpsR2 system, N and C terminal side deleting the terminal side, and the Q-linker domain PAS1 only at the downstream of the mutant PpsR2 using a (Q-PAS1) BphP1/Q-PAS1 systems have been developed (for example, Non-Patent Document 28-29) has.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • 発明者(英語)
  • IMAYOSHI Itaru
  • YAMADA Mayumi
  • SUZUKI Yusuke
  • NAGASAKI Shinji
国際特許分類(IPC)
指定国 National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
参考情報 (研究プロジェクト等) PRESTO Innovative technology platforms for integrated single cell analysis AREA
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