Production of ungulates, preferably bovines that produce human immunoglobulins

• US 20090276866A1
• Filed: 05/05/2008
• Published: 11/05/2009
• Est. Priority Date: 11/19/1999
• Status: Active Grant

• Current Assignee: SAB LLC (W.P. Carey, Inc. (Real Estate))
• Original Assignee: BioDak LLC
• Inventors: Kuroiwa, Yoshimi, Goldsby, Richard A., Osborne, Barbara A., Robl, James M.
• Granted Patent: US 7,820,878 B2
• Time in Patent Office: Days
• US Class Current: 800/15
• 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 ungulate wherein the expression of both copies of a gene essential for B-cell and T-cell production has been knocked out, selected from the group consisting of Igα

, E2A, EBF, BSAP, rag-1, and rag-2, which comprises the following steps;

(i) producing an ungulate cell wherein the expression of one or both copies of the Igα

, E2A, EBF, BSAP, rag-1, and/or rag-2 gene has been eliminated by targeted disruption;

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

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

(iv) obtaining a cloned fetus or animal ungulate that expresses the genotype of the donor differentiated cell, in which the expression of one or both copies of the Igα

, E2A, EBF, BSAP, rag-1, and/or rag-2 gene has been eliminated; and

(v) optionally, mating said male or female ungulate with another ungulate wherein one copy of the rag-1 or rag-2 gene has been knocked out and selecting progeny wherein both copies of the Igα

, E2A, EBF, BSAP, rag-1, or rag-2 genes have been knocked out.

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.

3. The method of claim 1, wherein the expression of both copies of the Igα

, E2A, EBF, BSAP, rag-1 and/or rag-2 gene is eliminated, by a three-step process comprising the following steps;

(i) producing an ungulate cell wherein the expression of one or both copies of the Igα

, E2A, EBF, BSAP, rag-1, and/or rag-2 gene has been eliminated by targeted disruption with a first DNA construct that provides for targeted disruption of said Igα

, E2A, EBF, BSAP, rag-1, and/or rag-2 gene;

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

(iii) contacting a 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 Igα

, E2A, EBF, BSAP, rag-1, and/or rag-2 gene by homologous recombination; and

(iv) fusing or inserting the resulting cell or nucleus therefrom, in which both copies of the Igα

, E2A, EBF, BSAP rag-1 and/or rag-2 gene have been knocked out, into an enucleated oocyte or blastomere, to produce an embryo which does not express Igα

, E2A, EBF, BSAP, rag-1, or rag-2.

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

5. The method of claim 1, wherein the ungulate cell used for homologous recombination is a differentiated cell derived from ectoderm, mesoderm or endoderm.

6. The method of claim 1, wherein the donor differentiated cell is a fibroblast cell.

7. The method of claim 1 or 2, wherein the cloned ungulate is selected from the group consisting of bovines, pigs, horses, sheep, buffalo, and goats.

8. The method of claim 7, wherein said cloned ungulate is a bovine.

9. The method of claim 1, wherein the differentiated cell of (i) is produced by sequentially contacting said cell with two knockout constructs which in combination provide for knockout of both copies of the Igα

, E2A, EBF, BSAP, rag-1, and/or rag-2 genes.

10. The method of claim 9, wherein the said two knockout constructs comprise different selectable markers thereby providing for the selection of cells wherein both copies of the rag-1 and/or rag-2 are eliminated.

11. The method of claim 10, wherein the two knockout constructs are pR3KOhyg and pR2KObsr.

12. The method of claim 1, wherein said method further comprises the step of introducing the double knockout embryo of (iv) into a female ungulate in order to produce a fetus or live offspring.

14. The transgenic ungulate according to claim 10 which is selected from the group consisting of bovine, pig, sheep, goat, horse, and buffalo.

15. The transgenic ungulate of claim 14 which is a bovine.

13. A transgenic ungulate wherein both copies of the rag-1 and/or rag-2 gene have been knocked out.