Nuclear transfer embryo formation method

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.

Description

RELATED APPLICATION [0001] This application is a continuation of International Application No. PCT / US03 / 23464, which designated the United States and was filed Jul. 29, 2003, published in English, which claims priority to and is a continuation-in-part of U.S. application Ser. No. 10 / 208,653, filed Jul. 29, 2002, the entire teachings of which are incorporated herein by reference.GOVERNMENT SUPPORT [0002] This invention was supported, in whole or in part, by Grant No. 2001-35205-09966 from the United States Department of Agriculture. The Government has certain rights in the invention.BACKGROUND OF THE INVENTION [0003] Nuclear transfer methods have been developed and used successfully to produce cloned sheep, cattle, mice, goats and pigs. Two cell components are combined to produce a cloned embryo; the donor nuclear genome (karyoplast) that is the target for clonal replication, and the enucleated oocyte (cytoplast) whose cytoplasmic constituency is sufficiently competent to facilitate ...

AI Technical Summary

Benefits of technology

[0017] The methods of the invention have advantages over methods of nuclear transfer that employ mechanical enucleation (e.g., enucleation using a micropipette to remove the nucleus) of the oocyte for several reasons. For example, using physical enucleation, the nucleus along with other important spindle associated elements are also removed. These spindle associated elements include, for example, centrosome factors (α-tubulin, pericentrin), kinases (e.g., C-mos, MPF, PAPK) and competence factors (e.g., cdc25C, spindlin). Several of these elements are important in forming a nuclear transfer embryo that is developmentally competent (e.g., embryo's ability or likelihood to develop successfully in vivo following an embryo transfer and implantation to a recipient mammal). The method of the invention removes the endogenous nucleus from the oocyte, while allowing several of these spindle associated elements to remain in the oocyte, thereby potentially significantly improving yield of competent embryos.

Problems solved by technology

However, physical enucleation is generally technically demanding, time consuming, inherently invasive and clearly damaging to cytoplast spatial organization.

Moreover, in certain instances, development of reconstructed embryos is inherently inefficient.

However, certain studies have reported that exposure of metaphase I and MII oocytes to etoposide, a topoisomerase II inhibitor, and cycloheximide yields enucleated cytoplasts with limited ability to support cleavage or blastocyst development, and term development of reconstructed embryos has not been reported.

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