# US 2009/0271884 A1 · ES Cell-Derived Mice From Diploid Host Embryo Injection

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- Number: US 2009/0271884 A1
- Document ID: US20090271884A1
- Status: Published application
- Kind: A1
- Application number: 12/399,109
- Filed: 2009-03-06
- Published: 2009-10-29
- Earliest claimed: 2008-03-07
- Assignees: Regeneron Pharmaceuticals, Inc.
- Inventors: William POUEYMIROU; Thomas M. DECHIARA; Wojtek AUERBACH; Aris N. ECONOMIDES; Nicholas W. GALE; David FRENDEWEY; David M. VALENZUELA
- Web page: https://patentgrep.com/patent/US20090271884A1

## CPC

- A01K 67/0271: Chimeric vertebrates, e.g. comprising exogenous cells
- A01K 67/0275: Genetically modified vertebrates, e.g. transgenic
- C12N 5/16: Animal cells
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- C12N 15/907: {in mammalian cells}
- A01K 2207/12: Animals modified by administration of exogenous cells
- A01K 2217/00: Genetically modified animals
- A01K 2217/05: Animals comprising random inserted nucleic acids (transgenic)
- A01K 2217/07: Animals genetically altered by homologous recombination
- A01K 2217/15: Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
- A01K 2217/203: Animal model comprising inducible/conditional expression system, e.g. hormones, tet
- A01K 2217/30: Animal model comprising expression system for selective cell killing, e.g. toxins, enzyme dependent prodrug therapy using ganciclovir
- A01K 2227/105: Murine
- A01K 2267/02: Animal zootechnically ameliorated
- C12N 2510/00: Genetically modified cells
- C12N 2517/02: Cells from transgenic animals
- 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)

## Abstract

Genetically modified mice and nucleic acid constructs for making the genetically modified mice are described. A first mouse having a gene encoding an activator (such as a Cre recombinase) operably linked to a developmentally-regulated promoter (such as a Nanog promoter) is provided. A second mouse having a toxic responder gene (such as a gene encoding diphtheria toxin A) is provided, where the toxic gene is expressed only in the presence of an activator, Embryos from a mating of the first and the second mouse are provided as host embryos suitable for generating mice from donor cells introduced into the host embryos. Ablating the ICM of a mouse embryo physically, chemically, or genetically is described, as well as making F0 generation mice that are substantially or in full derived from donor cells, employing a host mouse embryo with an ablated or nonproliferating ICM.

## Claims

### Claim 1

A mouse embryo, comprising a cell having in its genome:
(a) a site-specific recombinase gene operably linked to a developmentally-regulated promoter that expresses the site-specific recombinase gene in a cell of the inner cell mass (ICM) during an embryo stage; and,
(b) a gene whose expression prevents proliferation of an ICM cell, wherein expression of the gene whose expression prevents proliferation of the ICM cell is induced by the presence of the site-specific recombinase.

### Claim 2 (depends on 1)

The mouse embryo of claim 1, wherein the site-specific recombinase is a Cre recombinase or a modified Cre recombinase.

### Claim 3 (depends on 1)

The mouse embryo of claim 1, wherein the developmentally-regulated promoter is a Nanog promoter.

### Claim 4 (depends on 1)

The mouse embryo of claim 1, wherein the embryo stage is selected from a 2-cell stage, a 4-cell stage, an 8-cell stage, a 16-cell stage, and a 32-cell stage.

### Claim 5 (depends on 1)

The mouse embryo of claim 1, wherein the embryo stage is selected from a pre-morula, a morula, and a blastocyst.

### Claim 6 (depends on 1)

The mouse embryo of claim 1, wherein the gene whose expression prevents proliferation of an ICM cell is a gene encoding DTA.

### Claim 7 (depends on 1)

The mouse embryo of claim 1, wherein a site-specific recombinase recognition site flanks each end of a nucleic acid sequence that inhibits expression of the gene whose expression prevents proliferation of the ICM cell.

### Claim 8 (depends on 1)

The mouse embryo of claim 1, wherein the site-specific recombinase gene encodes a Cre recombinase, the developmentally-regulated promoter is a Nanog promoter, the gene whose expression prevents proliferation of the ICM cell is a gene encoding DTA, and the embryo stage is selected from a 2-cell stage, a 4-cell stage, an 8-cell stage, a 16-cell stage, and a 32-cell stage.

### Claim 9 (depends on 5)

The mouse embryo of claim 5, wherein the embryo is a blastocyst.

### Claim 10 (depends on 9)

The mouse embryo of claim 9, wherein the blastocyst substantially lacks a primitive endoderm.

### Claim 11

A method for making a mouse or mouse embryo from a mouse donor cell and a host embryo, comprising:
(a) introducing a mouse donor cell into a mouse host embryo, wherein the host embryo comprises
(i) a site-specific recombinase gene operably linked to a developmentally-regulated promoter that expresses the site-specific recombinase gene in a cell of the inner cell mass (ICM) during an embryo stage; and,
(ii) a gene whose expression prevents proliferation of an ICM cell, wherein expression of the gene whose expression prevents proliferation of the ICM cell is induced by the presence of the site-specific recombinase.
(b) introducing the mouse donor cell into the host embryo of step (a); and,
(c) gestating the embryo of step (b) in a pseudopregnant mouse.

### Claim 12 (depends on 11)

The method of claim 11, wherein the site-specific recombinase is a Cre recombinase or a modified Cre recombinase.

### Claim 13 (depends on 11)

The method of claim 11, wherein the developmentally-regulated promoter is a Nanog promoter.

### Claim 14 (depends on 11)

The method of claim 11, wherein the embryo stage is selected from a 2-cell stage, a 4-cell stage, an 8-cell stage, a 16-cell stage, and a 32-cell stage.

### Claim 15 (depends on 11)

The method of claim 11, wherein the embryo stage is selected from a pre-morula, a morula, and a blastocyst.

### Claim 16 (depends on 11)

The method of claim 11, wherein the gene whose expression prevents proliferation of an ICM cell is gene encoding DTA.

### Claim 17 (depends on 11)

The method of claim 11, wherein a site-specific recombinase recognition site flanks each end of a nucleic acid sequence that inhibits expression of the gene whose expression prevents proliferation of the ICM cell.

### Claim 18 (depends on 11)

The method of claim 11, wherein the site-specific recombinase gene encodes a Cre recombinase, the developmentally-regulated promoter is a Nanog promoter, the gene whose expression prevents proliferation of the ICM cell is a gene encoding DTA, and the embryo stage is selected from a 2-cell stage, a 4-cell stage, an 8-cell stage, a 16-cell stage, and a 32-cell stage.

### Claim 19 (depends on 18)

The method of claim 18, wherein the embryo is a blastocyst.

### Claim 20 (depends on 19)

The method of claim 19, wherein the blastocyst substantially lacks a primitive endoderm.

### Claim 21 (depends on 11)

The method of claim 11, wherein the donor cell is selected from an embryonic stem (ES) cell and an iPS cell.

### Claim 22 (depends on 11)

The method of claim 11, wherein following gestation in the pseudopregnant mouse, a mouse pup is born, wherein the mouse pup is fully derived from the donor cell.