Production of ungulates, preferably bovines that produce human immunoglobulins
First Claim
1. A method for producing a cloned ungulate wherein the expression of both copies of a gene essential for B cell production has been knocked out, selected from the group consisting of Igα
- , IgM, E2A, EBF, BSAP, rag-1 and rag-2, which comprises the following steps;
(i) producing a male and/or female ungulate cell wherein the expression of one or both copies of the Igα
, E2A, EBF, BSAP, IgM heavy chain, rag-1 and/or rag-2 gene has been eliminated by targeted disruption;
(ii) using said cell or DNA therefrom as a donor for nuclear transfer by fusing or inserting said donor cell or nucleus into an oocyte or blastomere, which is enucleated before or after transfer, activating the resulting nuclear transfer unit and/or the oocyte prior or simultaneous to nuclear transfer and culturing in a suitable medium to produce a nuclear transfer embryo;
(iii) introducing said nuclear transfer 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 one or both copies of the IgM (mu) chain, Igα
, E2A, EBF, BSAP, rag-1 and/or rag-2 gene have been eliminated; and
(v) optionally, mating said cloned male or female ungulate with another cloned female ungulate wherein one copy of the IgM, rag-1 or rag-2 gene has been knocked out and selecting progeny wherein both copies of the Igα
, E2A, EBF, BSAP, IgM, rag-1 or rag-2 genes have been knocked out.
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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.
110 Citations
32 Claims
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1. A method for producing a cloned ungulate wherein the expression of both copies of a gene essential for B cell production has been knocked out, selected from the group consisting of Igα
- , IgM, E2A, EBF, BSAP, rag-1 and rag-2, which comprises the following steps;
(i) producing a male and/or female ungulate cell wherein the expression of one or both copies of the Igα
, E2A, EBF, BSAP, IgM heavy chain, rag-1 and/or rag-2 gene has been eliminated by targeted disruption;
(ii) using said cell or DNA therefrom as a donor for nuclear transfer by fusing or inserting said donor cell or nucleus into an oocyte or blastomere, which is enucleated before or after transfer, activating the resulting nuclear transfer unit and/or the oocyte prior or simultaneous to nuclear transfer and culturing in a suitable medium to produce a nuclear transfer embryo;
(iii) introducing said nuclear transfer 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 one or both copies of the IgM (mu) chain, Igα
, E2A, EBF, BSAP, rag-1 and/or rag-2 gene have been eliminated; and
(v) optionally, mating said cloned male or female ungulate with another cloned female ungulate wherein one copy of the IgM, rag-1 or rag-2 gene has been knocked out and selecting progeny wherein both copies of the Igα
, E2A, EBF, BSAP, IgM, rag-1 or rag-2 genes have been knocked out. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 25, 26, 27, 28)
- , IgM, E2A, EBF, BSAP, rag-1 and rag-2, which comprises the following steps;
- 15. A transgenic ungulate wherein both copies of the IgM heavy chain (mu), rag-1 and/or rag-2 gene have been knocked out.
Specification