# US 2013/0160151 A1 · Transgenic animal as a model for identifying adult stem cells, and uses thereof

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- Number: US 2013/0160151 A1
- Document ID: US20130160151A1
- Status: Published application
- Kind: A1
- Application number: 13/808,343
- Filed: 2011-07-06
- Published: 2013-06-20
- Earliest claimed: 2010-07-07
- Assignees: Universite Pierre Et Marie Curie (Paris 6)
- Inventors: David Sassoon; Vanessa Besson; Giovanna Marazzi
- Web page: https://patentgrep.com/patent/US20130160151A1

## CPC

- C12Q 1/025: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18)}
- 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}
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- A01K 2217/07: Animals genetically altered by homologous recombination
- A01K 2227/105: Murine
- A01K 2267/0393: Animal model comprising a reporter system for screening tests
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to the use of a transgenic non-human animal, such as a mouse, expressing a reporter gene detectable by a chromogenic, luminescent or fluorescent signal which identifies the cells that express Pw1, or of Pw1-expressing cells or tissues isolated therefrom, as a model for screening a candidate substance for its ability to stimulate adult stem cells, or for monitoring cell aging.

## Claims

### Claim at position 1

-15. (canceled)

### Claim 16

An in vitro method for screening a candidate substance for its ability to stimulate adult stem cells, which method comprises the steps consisting of:
a. providing adult stem cells isolated from a transgenic non-human animal expressing a reporter gene detectable by a chromogenic, luminescent, or fluorescent signal which identifies the cells that express Pw1 that is a marker of adult stem cells;
b. contacting said stem cells with the candidate substance; and
c. determining the ability of the candidate substance to trigger, maintain or enhance the chromogenic, luminescent or fluorescent signal, whereby identifying the substance as being able to stimulate adult stem cells.

### Claim 17 (depends on 16)

The method of claim 16, wherein the stem cells of step (a) are isolated by an analysis of the chromogenic, luminescent or fluorescent signal of the reporter gene, optionally coupled to an analysis of stem cell marker(s), and wherein step (c) comprises determining the ability of the candidate substance to enhance the signal.

### Claim 18 (depends on 16)

The method of claim 16, wherein the stem cells of step (a) are isolated by an analysis of stem cell marker(s) without any chromogenic, luminescent or fluorescent signal being detectable, and wherein step (c) comprises determining the ability of the candidate substance to trigger said signal.

### Claim 19 (depends on 18)

The method of claim 18, wherein the stem cells of step (a) are isolated from a transgenic non-human animal that has been subjected to a stress, consisting preferably of hypoxia, NO-induced stress, H2O2-induced stress, thermal stress, or a chemically induced stress.

### Claim 20 (depends on 19)

The method of claim 19, wherein the animal has been subjected to a histone inhibitor.

### Claim 21 (depends on 19)

The method of claim 19, wherein the expression of the reporter gene is maternally transmitted and silenced in the non-human transgenic animal, and the candidate substance is screened for its ability to induce expression of said reporter gene.

### Claim 22 (depends on 16)

The method according to claim 16, wherein the stem cells of step (a) are isolated from a tissue selected from the group consisting of blood, bone marrow, hematopoietic system, skin, hair follicle, muscle, nervous system, heart, intestine, thymus, pancreas, testis, eye, kidney, liver, lung, spleen, tongue, bones and dental pulp.

### Claim 23 (depends on 16)

The method according to claim 16, wherein the transgenic non-human animal has been generated or is a progeny of a non-human animal generated by introduction, into a non-human animal ovocyte, of a BAC recombinant vector comprising a reporter gene inserted into a PW1 gene.

### Claim 24 (depends on 16)

The method according to claim 16, wherein the reporter gene encodes β-galactosidase or Green Fluorescent Protein.

### Claim 25 (depends on 16)

The method of claim 16, wherein the non-human animal is a mammal.

### Claim 26 (depends on 25)

The method of claim 25, wherein the non-human animal is a rodent.

### Claim 27 (depends on 26)

The method of claim 26, wherein the non-human animal is a mouse.

### Claim 28

An in vivo method for screening a candidate substance for its ability to stimulate adult stem cells, which method comprises administering a transgenic non-human animal expressing a reporter gene detectable by a chromogenic, luminescent or fluorescent signal which identifies the cells that express Pw1, that is a marker of adult stem cells, with the candidate substance; and determining the ability of the candidate substance to trigger, maintain or enhance the chromogenic, luminescent or fluorescent signal, whereby identifying the substance as being able to stimulate adult stem cells.

### Claim 29 (depends on 28)

The method according to claim 28, wherein the transgenic non-human animal has been generated or is a progeny of a non-human animal generated by introduction, into a non-human animal ovocyte, of a BAC recombinant vector comprising a reporter gene inserted into a PW1 gene.

### Claim 30 (depends on 28)

The method according to claim 28, wherein the reporter gene encodes β-galactosidase or Green Fluorescent Protein.

### Claim 31 (depends on 28)

The method of claim 28, wherein the non-human animal is a mammal.

### Claim 32 (depends on 31)

The method of claim 31, wherein the non-human animal is a rodent.

### Claim 33 (depends on 32)

The method of claim 32, wherein the non-human animal is a mouse.

### Claim 34

A method for cultivating non-human animal adult stem cells, which method comprises the steps consisting of:
a. providing a transgenic non-human animal expressing a reporter gene detectable by a chromogenic, luminescent or fluorescent signal which identifies the cells that express Pw1 that is a marker of adult stem cells;
b. isolating adult stem cells from the transgenic non-human animal by analyzing the chromogenic, luminescent or fluorescent signal of the reporter gene, optionally coupled to an analysis of stem cell marker(s); and
c. allowing the isolated adult stem cells to grow in vitro,

### Claim 35 (depends on 34)

The method according to claim 34, wherein the transgenic non-human animal has been generated or is a progeny of a non-human animal generated by introduction, into a non-human animal ovocyte, of a BAC recombinant vector comprising a reporter gene inserted into a PW1 gene.

### Claim 36 (depends on 34)

The method according to claim 34, wherein the reporter gene encodes β-galactosidase or Green Fluorescent Protein.

### Claim 37 (depends on 34)

The method of claim 34, wherein the non-human animal is a mammal.

### Claim 38 (depends on 37)

The method of claim 37, wherein the non-human animal is a rodent.

### Claim 39 (depends on 38)

The method of claim 38, wherein the non-human animal is a mouse.

### Claim 40

A method for screening a candidate substance for its ability to stimulate adult stem cells, which method uses a transgenic non-human animal model expressing a reporter gene detectable by a chromogenic, luminescent or fluorescent signal which identifies the cells that express Pw1, or of Pw1 -expressing cells or tissues isolated therefrom.

### Claim 41 (depends on 40)

The method of claim 40, wherein the candidate substance is a chemical drug, a biological compound, or a mixture of natural compounds.

### Claim 42 (depends on 40)

The method of claim 40, wherein the non-human animal is a mammal.

### Claim 43 (depends on 41)

The method of claim 41, wherein the non-human animal is a rodent.

### Claim 44 (depends on 42)

The method of claim 42, wherein the non-human animal is a mouse.

### Claim 45

A method for monitoring cell aging, which method uses a transgenic non-human animal model expressing a reporter gene detectable by a chromogenic, luminescent or fluorescent signal which identifies the cells that express Pw1, or of Pw1 -expressing cells or tissues isolated therefrom.

### Claim 46 (depends on 45)

The method of claim 45, for monitoring aging of adult stem cells present in skin or hair follicle.

### Claim 47 (depends on 45)

The method of claim 45,wherein the non-human animal is a mammal.

### Claim 48 (depends on 47)

The method of claim 47, wherein the non-human animal is a rodent.

### Claim 49 (depends on 48)

The method of claim 48, wherein the non-human animal is a mouse.