# US 10,765,094 B2 · Inflammation reporter system

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- Number: US 10,765,094 B2
- Document ID: US10765094B2
- Status: Granted
- Kind: B2
- Application number: 15/329,477
- Filed: 2014-07-31
- Published: 2017-08-03
- Granted: 2020-09-08
- Earliest claimed: 2014-07-31
- Assignees: Transgenic Inc.; National University Corporation Kumamoto University; National University Corporation Gunma University
- Applicants: Transgenic Inc.; National University Corporation Kumamoto University; National University Corporation Gunma University
- Inventors: Kenichi Yamamura; Takao Iwawaki; Daisuke Oikawa; Tomoo Ishikawa
- Web page: https://patentgrep.com/patent/US10765094B2

## CPC

- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- A01K 67/027: New or modified breeds of vertebrates
- A01K 67/0275: Genetically modified vertebrates, e.g. transgenic
- A61K 49/0008: {Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure}
- C07K 14/545: IL-1
- C12N 15/09: Recombinant DNA-technology
- A01K 2217/052: inducing gain of function
- A01K 2217/20: Animal model comprising regulated expression system
- A01K 2227/105: Murine
- A01K 2267/0368: Animal model for inflammation
- C07K 2319/61: containing an enzyme fusion for detection (lacZ, luciferase)
- C07K 2319/95: containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present invention provides a method for detection of an inflammatory reaction, which comprises using a transformant or transgenic non-human animal transfected with a vector comprising a promoter for a gene encoding an inflammatory cytokine, a gene encoding a reporter protein, a gene encoding the inflammatory cytokine, and a gene encoding a proteolytic signal sequence to thereby detect an inflammatory reaction induced upon inflammatory stimulation in the transformant or in the transgenic non-human animal.

## Claims

### Claim 1

A vector comprising an inflammatory cytokine promoter operably linked to a nucleotide sequence comprising a gene encoding a luciferase reporter protein, a gene encoding an inflammatory cytokine, wherein the inflammatory cytokine is a mouse-derived IL-1β partial sequence consisting of amino acids 17-216, and a gene encoding a proteolytic signal sequence,
wherein the luciferase reporter protein, the inflammatory cytokine and the proteolytic signal sequence are configured as a fusion protein, wherein the inflammatory cytokine is N-terminal to the proteolytic signal sequence and C-terminal to the luciferase reporter protein, and
wherein, in the presence of inflammatory stimulation, the inflammatory cytokine promoter becomes functional to thereby activate the expression of the nucleotide sequence and the inflammatory cytokine is cleavable by caspase-1 to separate the luciferase reporter protein from the proteolytic signal sequence while retaining luciferase activity.

### Claim 2 (depends on 1)

A non-human transformant comprising the vector according to claim 1.

### Claim 3 (depends on 1)

A transgenic non-human animal comprising in its genome the vector according to claim 1.

### Claim 4 (depends on 3)

The transgenic non-human animal according to claim 3, wherein the non-human animal is a mouse.

### Claim 5 (depends on 2)

The non-human transformant according to claim 2, wherein the reporter protein is detected as a luminescence signal upon inflammatory stimulation.

### Claim 6 (depends on 2, 3)

A method for detection of an inflammatory reaction, which comprises using the transformant according to claim 2 or the transgenic non-human animal according to claim 3 to detect an inflammatory reaction induced upon inflammatory stimulation in the transformant or in the transgenic non-human animal.

### Claim 7 (depends on 6)

The method according to claim 6, wherein the gene encoding the inflammatory cytokine is expressed by transcription factor NF-κB induced during inflammatory reaction.

### Claim 8 (depends on 6)

The method according to claim 6, wherein the reporter protein is detected as a luminescence signal upon inflammatory stimulation.

### Claim 9 (depends on 2, 3)

A method for screening of anti-inflammatory substances, which comprises bringing the transformant according to claim 2 or the transgenic non-human animal according to claim 3 into contact with candidate substances under inflammatory stimulation to select an anti-inflammatory substance on the basis of the presence or absence of an inflammatory reaction serving as an indicator.

### Claim 10 (depends on 2)

A kit for detection of an inflammatory reaction or for screening of anti-inflammatory substances, which comprises the non-human transformant according to claim 2.

### Claim 11 (depends on 1)

The vector of claim 1, wherein the promoter is an interleukin 1β inflammatory cytokine promoter, the reporter protein is photinus pyralis luciferase, and the proteolytic signal sequence comprises a CL1-PEST proteolytic signal sequence.