Cloning using donor nuclei from differentiated fetal and adult cells

Abstract

An improved method of nuclear transfer involving the transplantation of donor differentiated cell nuclei into enucleated oocytes of the same species as the donor cell is provided. The resultant nuclear transfer units are useful for multiplication of genotypes and transgenic genotypes by the production of fetuses and offspring, and for production of isogenic CICM cells, including human isogenic embryonic or stem cells. Production of genetically engineered or transgenic mammalian embryos, fetuses and offspring is facilitated by the present method since the differentiated cell source of the donor nuclei can be genetically modified and clonally propagated.

Description

[0001] This application is a continuation of U.S. Ser. No. 08 / 935,052, filed Sep. 22, 1997, which is a divisional of U.S. Ser. No. 08 / 781,752, filed Jan. 10, 1997, and which are incorporated herein in their entirety by reference.[0002] The present invention relates to cloning procedures in which cell nuclei derived from differentiated fetal or adult, mammalian cells are transplanted into enucleated mammalian oocytes of the same species as the donor nuclei. The nuclei are reprogrammed to direct the development of cloned embryos, which can then be transferred into recipient females to produce fetuses and offspring, or used to produce cultured inner cell mass cells (CICM). The cloned embryos can also be combined with fertilized embryos to produce chimeric embryos, fetuses and / or offspring.[0003] Methods for deriving embryonic stem (ES) cell lines in vitro from early preimplantation mouse embryos are well known. (See, e.g., Evans et al., Nature, 29:154-156 (1981); Martin, Proc. Natl. Ac...

AI Technical Summary

Benefits of technology

[0058] Thus, according to the present invention, multiplication of superior genotypes of mammals, including ungulates, is possible. This will allow the multiplication of adult animals with proven genetic superiority or other desirable traits. Progress will be accelerated, for example, in many important ungulate species. By the present invention, there are potentially billions of fetal or adult cells that can be harvested and used in the cloning procedure. This will potentially result in many identical offspring in a short period.

[0132] The great advantage of the subject invention is that it provides an essentially limitless supply of isogenic or syngenic human cells suitable for transplantation. Therefore, it will obviate the significant problem associated with current transplantation methods, i.e., rejection of the transplanted tissue which may occur because of host-vs-graft or graft-vs-host rejection. Conventionally, rejection is prevented or reduced by the administration of anti-rejection drugs such as cyclosporine. However, such drugs have significant adverse side-effects, e.g., immunosuppression, carcinogenic properties, as well as being very expensive. The present invention should eliminate, or at least greatly reduce, the need for anti-rejection drugs.

Problems solved by technology

However, there was no demonstration of development past early embryonic stages (blastocyst stage).

Also, granulosa cells are not easily cultured and are only obtainable from females.

While multiplications of genotypes are possible using embryonic cells as donors, there are problems with current methods.

There also exist problems in the area of producing transgenic mammals.

However, many early embryos are required to produce one transgenic animal and, thus, this procedure is very inefficient.

Also, there is no simple and efficient method of selecting for a transgenic embryo before going through the time and expense of putting the embryos into surrogate females.

In addition, gene targeting techniques cannot be easily accomplished with early embryo transgenic procedures.

Even if these precautions are followed, these cells often undergo spontaneous differentiation and cannot be used to produce transgenic offspring by currently available methods.

Also, some embryonic cell lines have to be propagated in a way that is not conducive to gene targeting procedures.