Embryonic or stem-like cell lines produced by cross species nuclear transplantation and methods for enhancing embryonic development by genetic alteration of donor cells or by tissue culture conditions

• US 7,696,404 B2
• Filed: 12/27/2002
• Issued: 04/13/2010
• Est. Priority Date: 08/19/1996
• Status: Expired due to Fees

• Current Assignee: Advanced Cell Technology Incorporated (Astellas Pharma Incorporated)
• Original Assignee: Advanced Cell Technology Incorporated (Astellas Pharma Incorporated)
• Inventors: West, Michael D., Cibelli, Jose
• Primary Examiner(s): Ton; Thaian N
• Assistant Examiner(s): NOBLE, MARCIA STEPHENS
• Application Number:
  US10/329,979
• Publication Number:
  US 20030229908A1
• Time in Patent Office: 2,664 Days
• Field of Search: 435/377, 435/366, 435/325, 800/24
• US Class Current: 800/24
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• CPC Class Codes: C12N 15/877 Techniques for producing new mammalian cloned embryos
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2517/04 Cells produced using nuclear transfer

Abstract

An improved method of nuclear transfer involving the transplantation of differentiated donor cell nuclei into enucleated oocytes of a species different from the donor cell is provided. The resultant nuclear transfer units are useful for the production of isogenic embryonic stem cells, in particular human isogenic embryonic or stem cells. These embryonic or stem-like cells are useful for producing desired differentiated cells and for introduction, removal or modification, of desired genes, e.g., at specific sites of the genome of such cells by homologous recombination. These cells, which may contain a heterologous gene, are especially useful in cell transplantation therapies and for in vitro study of cell differentiation. Also, methods for improving nuclear transfer efficiency by genetically altering donor cells to inhibit apoptosis, select for a specific cell cycle and/or enhance embryonic growth and development are provided.

Claims

1. A method for producing a nuclear transfer human/rabbit blastocyst by cross-species nuclear transfer to give rise to embryonic stem-like cells comprising:

(i) inserting a human cell or human cell nucleus into an enucleated recipient rabbit oocyte under conditions suitable for the formation of a nuclear transfer (NT) unit;

(ii) activating the NT unit formed in step (i); and

(iii) culturing the NT unit until the blastocyst stage to give rise to embryonic stem-like cells, wherein the embryonic stem-like cells are pluripotent.

2. The method of claim 1 which further comprises transferring the nucleus of the NT unit from step (i) into another rabbit oocyte which is enucleated before transfer of the nucleus.

3. The method of claim 1 wherein the nuclear transfer blastocyst produced by step (iii) is cultured on a feeder layer.

4. The method of claim 3 wherein the blastocyst is cultured on a fibroblast feeder layer.