# US 7,078,585 B1 · Bacterial cell component-unresponsive model mouse

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- Number: US 7,078,585 B1
- Document ID: US7078585B1
- Status: Granted
- Kind: B1
- Application number: 09/889,324
- Filed: 2000-01-13
- Granted: 2006-07-18
- Earliest claimed: 1999-01-14
- Assignees: Japan Science and Technology Agency
- Inventors: Shizuo Akira; Osamu Takeuchi; Kiyoshi Takeda
- Web page: https://patentgrep.com/patent/US7078585B1

## CPC

- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- A01K 67/0276: Knock-out vertebrates
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- G01N 33/5047: {Cells of the immune system}
- G01N 33/5055: {involving macrophages}
- A01K 2217/075: inducing loss of function, i.e. knock out
- A01K 2227/105: Murine
- A01K 2267/03: Animal model, e.g. for test or diseases
- A01K 2267/0337: Animal models for infectious diseases
- G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

A knockout mouse which is unresponsive to peptidoglycan, a lipoprotein/lipopeptide and the like, and is useful for elucidating the contribution of individual members of the TLR family to a signaling stimulated with bacterial cell components in vivo, in particular, the role of TLR2 and MyD88 in vivo. A bacterial cell component-unresponsive knockout mouse is generated by a process comprising the steps of: a targeting vector is constructed by replacing a whole or a part of a gene fragment of an exon region containing a cytoplasmic region of TLR2 or MyD88 gene and the like with a plasmid having a poly A signal and a marker gene; the targeting vector is introduced into an embryonic stem cell; the targeting embryonic stem cell having a homologously recombined TLR2 or MyD88 gene is microinjected into the blastocyst of a mouse and the blastocyst is put back into the uterus of a recipient mouse.

## Claims

### Claim 1

A mouse comprising homozygous disruption of TLR2 gene in its genome, wherein such disruption results in no production of endogenous TLR2 protein, and wherein said mouse exhibits the phenotype of being unresponsive to bacterial cell component(s) that is a lipoprotein/lipopeptide.

### Claim 2 (depends on 1)

The mouse according to claim 1, wherein a lipoprotein/lipopeptide is a macrophage-activating lipopeptide obtained from bacteria which belong to Mycoplasma.

### Claim 3 (depends on 1)

The mouse according to claim 1 that is further unresponsive to peptidoglycan as a bacterial cell component.

### Claim 4 (depends on 1)

The mouse according to claim 1 that is further hyporesponsive to a cell wall fraction of Gram-positive bacteria.

### Claim 5

A mouse comprising homozygous disruption of MyD88 gene in its genome, wherein such disruption results in no production of endogenous MyD88 protein, and wherein said mouse exhibits the phenotype of being unresponsive to bacterial cell component(s) that is a lipoprotein/lipopeptide.

### Claim 6 (depends on 5)

The mouse according to claim 5, wherein a lipoprotein/lipopeptide is a macrophage-activating lipopeptide obtained from bacteria which belong to Mycoplasma.

### Claim 7 (depends on 5)

The mouse according to claim 5 that is further unresponsive to peptidoglycan as a bacterial cell component.

### Claim 8 (depends on 5)

The mouse according to claim 5 that is further unresponsive to a cell wall fraction of Gram-positive bacteria.

### Claim 9 (depends on 5)

The mouse according to claim 5 that is further unresponsive to endotoxin as a bacterial cell component.

### Claim 10 (depends on 5)

The mouse according to claim 5 that is further unresponsive to lipoteichoic acid as a bacterial cell component.

### Claim 11 (depends on 5)

The mouse according to claim 5 that is further unresponsive to Mycobacterium tuberculosis lysate as a bacterial cell component.