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Atp-visualizing animal and use thereof UPDATE

外国特許コード F180009592
整理番号 5558
掲載日 2018年11月16日
出願国 アメリカ合衆国
出願番号 201515111730
公報番号 20180110880
出願日 平成27年1月15日(2015.1.15)
公報発行日 平成30年4月26日(2018.4.26)
国際出願番号 JP2015050932
国際公開番号 WO2015108102
国際出願日 平成27年1月15日(2015.1.15)
国際公開日 平成27年7月23日(2015.7.23)
優先権データ
  • 特願2014-005429 (2014.1.15) JP
  • 2015JP50932 (2015.1.15) WO
発明の名称 (英語) Atp-visualizing animal and use thereof UPDATE
発明の概要(英語) Provided are a transgenic non-human mammal expressing a fusion protein, wherein the fusion protein comprises an ε subunit of an ATP synthase and two distinct fluorescent proteins as a donor and an acceptor for FRET, one of the fluorescent proteins being placed at an amino terminal moiety of the ε subunit and the other being placed at a carboxyl terminal moiety of the ε subunit, and a method of screening for an agent for preventing or treating diseases in a mammal in need thereof, comprising using an above transgenic non-human mammal.
従来技術、競合技術の概要(英語) BACKGROUND ART
Adenosine triphosphate (ATP) is used in many biological reactions and called “cellular energy currency”.Monitoring ATP levels is accordingly important to assess the energy status in a living body.Methods conventionally and widely used to measure cellular ATP levels include a luciferase assay that includes the steps of: obtaining cells from an animal; measuring the weight of the cells or counting the number of the cells; homogenizing the cells; adding to the cell homogenate a composition containing luciferase as a main component; measuring the luminescence intensity; and estimating the ATP level.Luciferase assays are unsuitable for measuring cellular ATP levels precisely for the following reasons: the assays are highly sensitive while cause large measurement errors; the luminescence intensity varies depending on time after the addition of the composition comprising luciferase, the oxygen concentration and the pH of the solution to be measured; the precise number of the cells can hardly be estimated; and the assay result varies depending on procedures for homogenizing the cells.In addition, homogenization of a cell destroys its subcellular compartments including cytoplasm and cell organelles such as mitochondria, thereby being unable to measure the ATP level at each of the cellular compartments or the time-dependent ATP levels.
Other methods for measuring ATP levels in cells or organs include a mass spectroscopy assay, which comprises the steps of: pre-treating the cells or organs; and measuring the ATP level with a mass spectroscopy detector.Mass spectroscopy also have some problems: the assay requires a large number of cells and can hardly be performed with a single cell; the pre-treatment such as homogenization of the cells leads large measurement errors and destroys the subcellular compartments including cytoplasm and cell organelles such as mitochondria, thereby being unable to measure the ATP level at each of subcellular compartments or the time-dependent ATP levels.
Methods using fluorescence resonance energy transfer (FRET) have been recently reported (non-patent literature 1).The method utilizes a fusion protein that includes a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) which are fused to the CBS domain of inosine monophosphate dehydrogenase 2 (IMPDH2).IMPDH2 can react with adenosine nucleic acids such as ATP, adenosine diphosphate (ADP) and adenosine monophosphate (AMP).Patent literature 1 discloses a method for measuring the ATP level using FRET which utilizes a fluorescence-labeled fusion protein including an ε protein derived from an ATP synthase F0F1 ATP-sensitive peptide of Bacillus subtilis and two distinct fluorescent proteins, which are connected to the εprotein.The two fluorescent proteins act as a donor and an acceptor for FRET.However, it still remains difficult to quantitatively measure the time-dependent level or distribution of ATP in a single living cell.
特許請求の範囲(英語) [claim1]
1. A transgenic non-human mammal expressing a fusion protein, wherein
the fusion protein comprises an ε subunit of an ATP synthase or a fragment thereof and two distinct fluorescent proteins as a donor and an acceptor for fluorescence resonance energy transfer (FRET);
the transgenic non-human mammal has a DNA encoding the fusion protein which is inserted into the ROSA26 locus on the chromosome of the mammal; and
the DNA encoding the fusion protein is operably linked to a CAG promoter sequence.
[claim2]
2. The transgenic non-human mammal according to claim 1, wherein the ε subunit comprises an amino acid sequence of any one of SEQ ID NOs: 1-3 or a variant sequence having substitution, insertion, or deletion of one to five amino acid(s) in the sequence of any one of SEQ ID NOs: 1-3.
[claim3]
3. The transgenic non-human mammal according to claim 1, wherein the two distinct fluorescent proteins are a combination selected from the group consisting of a cyan fluorescent protein (CFP), a yellow fluorescent protein (YFP), a blue fluorescent protein (BFP), a green fluorescent protein (GFP), a red fluorescent protein (RFP) and an orange fluorescent protein (OFP).
[claim4]
4. (canceled)
[claim5]
5. The transgenic non-human mammal according to claim 1, wherein the DNA encoding the fusion protein comprises target sequences and a Stop sequence sandwiched between the target sequences; and the fusion protein is expressed when the Stop sequence sandwiched between the target sequences is removed with a recombinase which recognizes the target sequences.
[claim6]
6. (canceled)
[claim7]
7. A transgenic non-human mammal expressing a fusion protein, which is produced by mating the transgenic non-human mammal expressing the fusion protein according to claim 1 with a disease model animal.
[claim8]
8. The transgenic non-human mammal according to claim 7, wherein the disease model animal is an autism-model animal a diabetic-model animal, a cancer-model animal, an infection-model animal, or a heart disease-model animal.
[claim9]
9. The transgenic non-human mammal according to claim 1, being wherein the mammal is a mouse.
[claim10]
10. A method of evaluating the efficacy of a test substance to alter an ATP level, comprising the steps of:
contacting the transgenic non-human mammal expressing the fusion protein according to claim 1, or a cell, an organ or a tissue of the transgenic non-human mammal with the test substance,
measuring fluorescence emission arising from the fusion protein expressed, and
evaluating the efficacy of the test substance to alter the ATP level by using the measured fluorescence emission.
[claim11]
11. (canceled)
[claim12]
12. The transgenic non-human mammal according to claim 1, wherein the ε subunit or the fragment thereof comprises the N- and C-terminal domains, the N-terminal domain forms plural beta-strands, and the C-terminal domain forms double alpha-helices.
[claim13]
13. The transgenic non-human mammal according to claim 1, wherein one of the two distinct fluorescent proteins is placed at the amino terminal moiety of the ε subunit and the other is placed at the carboxyl terminal moiety of the ε subunit.
[claim14]
14. The transgenic non-human mammal according to claim 1, being a mouse, a rat, a rabbit, a goat, a pig, a dog, a cat, a guinea pig, a hamster, a sheep, a cow, or a marmoset.
[claim15]
15. The transgenic non-human mammal according to claim 5, wherein the target sequences are selected from loxP, lox71, lox66, lox511,lox2272, Vlox (VCre), Slox (SCre) and FRT sequence.
[claim16]
16. The transgenic non-human mammal according to claim 5, wherein the Stop sequence comprises a SV40pA sequence.
[claim17]
17. The method according to claim 10, wherein the evaluating step comprises a step of comparing the fluorescence emission measured in the measuring step with the fluorescence emission measured prior to the contacting step.
  • 発明者/出願人(英語)
  • YAMAMOTO Masamichi
  • KYOTO UNIVERSITY
国際特許分類(IPC)
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