# US 7,612,250 B2 · Nuclear transfer embryo formation method

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- Number: US 7,612,250 B2
- Document ID: US7612250B2
- Status: Granted
- Kind: B2
- Application number: 10/208,653
- Filed: 2002-07-29
- Published: 2004-01-29
- Granted: 2009-11-03
- Earliest claimed: 2002-07-29
- Assignees: Trustees of Tufts College
- Inventors: Eric W. Overstrom; Daniela Fischer Russell
- Web page: https://patentgrep.com/patent/US7612250B2

## CPC

- C12N 15/8771: {Bovine embryos}
- C12N 15/873: Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
- A01K 2227/101: Bovine
- 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

## Abstract

A nuclear transfer embryo is formed by destabilizing microtubules of an oocyte, whereby essentially all endogenous chromatin collects at a second polar body during meiosis of an oocyte. The oocyte is fused with the nucleus of a donor somatic cell of the same species of said oocyte prior to cessation of extrusion of the second polar body from the oocyte, thereby forming the nuclear transfer embryo. In one embodiment, the nuclear transfer embryo is employed to impregnate an animal, such as a mammal. In another embodiment, the donor nucleus is transgenic.

## Claims

### Claim 1

A method of forming a nuclear transfer embryo, comprising the steps of:
a) destabilizing microtubules of an oocyte, whereby all endogenous nuclear chromatin collects at a second polar body during meiosis of said oocyte; and
b) combining the oocyte with at least the nucleus of a donor cell of the same species of said oocyte before a protrusion of the second polar body containing all of the endogenous nuclear chromatin ceases, thereby forming a nuclear transfer embryo containing the nucleus of the donor cells wherein the endogenous nuclear chromatin of the oocyte is in the second polar body.

### Claim 2 (depends on 1)

The method of claim 1, wherein the microtubules are destabilized by combining the oocyte with a chemical selected from the group consisting of demecolcine, paclitaxel, phalloidin, coichicine, and nocodozole.

### Claim 3 (depends on 2)

The method of claim 2, further includes activating the oocyte prior to exposing the oocyte to said chemical.

### Claim 4 (depends on 1)

The method of claim 1, wherein the microtubules are destabilized by exposing the microtubules to electromagnetic radiation.

### Claim 5 (depends on 4)

The method of claim 4, wherein the electromagnetic radiation is selected from the group consisting of x-rays and heat.

### Claim 6 (depends on 1)

The method of claim 1, wherein the microtubules are destabilized by exposure of the oocyte to a change in pH or osmolality.

### Claim 7 (depends on 1)

The method of claim 1, wherein the oocyte is in a telophase II or anaphase II stage of meiosis.

### Claim 8 (depends on 1)

The method of claim 1, wherein the oocyte is in a metaphase II stage of meiosis.

### Claim 9 (depends on 8)

The method of claim 8, further including the step of activating the oocyte.

### Claim 10 (depends on 1)

The method of claim 1, wherein the donor cell is an activated donor cell.

### Claim 11 (depends on 10)

The method of claim 10, wherein the activated donor cell is in the G1 stage of a mitotic cell cycle.

### Claim 12 (depends on 11)

The method of claim 11, wherein the activated donor cell is a fibroblast activated donor cell.

### Claim 13 (depends on 12)

The method of claim 12, wherein the somatic activated donor cell is an epithelial activated donor cell.

### Claim 14 (depends on 1)

The method of claim 1, wherein the donor cell is a somatic cell.

### Claim 15 (depends on 14)

The method of claim 14, wherein the donor cell is an adult somatic cell.

### Claim 16 (depends on 14)

The method of claim 14, wherein the donor cell is an embryonic somatic cell.

### Claim 17 (depends on 1)

The method of claim 1, wherein the oocyte is mammalian.

### Claim 18 (depends on 1)

The method of claim 1, wherein the donor cell is transgenic.

### Claim 19 (depends on 10)

The method of claim 10, wherein the compound that destabilizes the microtubules is selected from the group consisting of demecolcine, nocodazole, colchicine, phalloidin and paclitaxel.

### Claim 20

A method of forming a non-human, mammalian nuclear transfer embryo, comprising the steps of:
a) destabilizing microtubules of an oocyte, whereby all endogenous nuclear genetic material collects at a second polar body during meiosis of said oocyte; and
b) combining the oocyte with at least the nucleus of a donor somatic cell of the same species of said oocyte before protrusion of the second polar body containing all of the endogenous nuclear chromatin ceases, thereby forming a nuclear transfer embryo.