Production of ungulates, preferably bovines that produce human immunoglobulins
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.
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.
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.
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).
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.