Production of ungulates, preferably bovines that produce human immunoglobulins

• US 7,820,878 B2
• Filed: 05/05/2008
• Issued: 10/26/2010
• Est. Priority Date: 11/19/1999
• Status: Expired due to Term

• Current Assignee: SAB LLC (W.P. Carey, Inc. (Real Estate))
• Original Assignee: Kyowa Hakko Kirin Company Limited (Kirin Holdings Company Limited)
• Inventors: Kuroiwa, Yoshimi, Goldsby, Richard A., Osborne, Barbara A., Robl, James M.
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US12/151,181
• Publication Number:
  US 20090276866A1
• Time in Patent Office: 904 Days
• Field of Search: 800/24
• US Class Current: 800/24
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/052 inducing gain of function
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/101 Bovine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)

Abstract

The present invention relates to a method of producing an ungulate having both copies of the IgM heavy chain (mu) rag-1 and/or rag-2 gene eliminated from its genome. Animals which have IgM, rag-1 and/or rag-2 eliminated from their genome are unable to conduct the gene rearrangements that are necessary to generate the antigen receptors of B- or T-lymphocytes, and therefore will not develop native B- or T-cells. Because they are unable to produce B- and T-lymphocytes, these IgM, rag-1, or rag-2 ungulates cannot reject human hematopoietic stem cell preparations, and B- and T-lymphocytes which develop therefrom. Therefore, the present invention also involves injecting into IgM, rag-1, and/or rag-2 deficient ungulates, in utero or shortly after birth, human B- and T-lymphocytes whose immune systems produce human immunoglobulin that can be processed for therapeutic uses in humans.

Claims

1. A method for producing a cloned bovine, wherein the expression of both copies of the rag-2 gene have been knocked out and wherein said bovine lacks viable B- and T-cells, which comprises the following steps:

(i) producing a donor bovine fibroblast cell or nucleus therefrom wherein the expression of one or both copies of the rag-2 gene has been eliminated by targeted disruption;

(ii) fusing or inserting said donor fibroblast cell or nucleus into an enucleated bovine oocyte, to produce an embryo;

(iii) introducing said embryo into a female bovine; and

(iv) obtaining a cloned fetus or bovine animal that expresses the genotype of the donor fibroblast cell, in which the expression of one or both copies of the rag-2 gene has been eliminated; and

(v) optionally, mating said bovine animal with another bovine animal wherein one copy of the rag-2 gene has been knocked out and selecting progeny wherein both copies of the rag-2 gene have been knocked out resulting in a lack of viable V-and T-cells in said bovine animal.

2. The method of claim 1, wherein said one or both copies of the rag-2 gene has been eliminated by targeted disruption with pR3KOhyg or pR2KObsr.

5. The method of claim 1, wherein the fibroblast cell of step (i) is produced by sequentially contacting said cell with two knockout constructs which in combination provide for knockout of both copies of the rag-2 gene.

6. The method of claim 5, wherein the said two knockout constructs comprise different selectable markers thereby providing for the selection of fibroblasts wherein both copies of the rag-2 gene are eliminated.

7. The method of claim 6, wherein the two knockout constructs are pR3KOhyg and pR2KObsr.

8. The method of claim 1, wherein both copies of the rag-2 gene have been eliminated in the embryo of step (ii).

3. A method for producing a cloned bovine, wherein the expression of both copies of the rag-2 gene have been knocked out by a three-step process which comprises the following steps:

(i) producing a donor bovine fibroblast an cell wherein the expression of one copy of the rag-2 gene has been eliminated by targeted disruption with a first DNA construct that provides for targeted disruption of said rag-2 gene;

(ii) fusing or inserting said donor fibroblast cell or nucleus therefrom into an enucleated bovine oocyte to produce an embryo;

(iii) contacting a fibroblast cell from said embryo with a second DNA construct under conditions that result in the elimination of the expression of the second copy of the rag-2 gene by homologous recombination; and

(iv) fusing or inserting the resulting fibroblast cell or nucleus therefrom, in which both copies of the rag-2 gene have been knocked out, into an enucleated bovine oocyte or blastomere, to produce an embryo which does not express rag-2, resulting in a lack of viable B-and T-cells in said bovine animal.

4. The method of claim 3, wherein the first DNA construct is pR3KOhyg and the second DNA construct is pR2KObsr.

9. A transgenic bovine wherein both copies of the rag-2 gene have been knocked out resulting in a lack of viable B-and T-cells in said bovine animal.