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  Pharmaceutical Patents  

 

Title:  Transgenic bovines having reduced prion protein production
United States Patent: 
7,429,690
Issued: 
September 30, 2008

Inventors: 
Robl; James M. (Brandon, SD), Kuroiwa; Yoshimi (Sioux Falls, SD), Tomizuka; Kazuma (Takasaki, JP), Ishida; Isao (Isehara, JP)
Assignee: 
Kirin Holdings Kabushiki Kaisha (Tokyo, JP)
Appl. No.: 
10/705,519
Filed: 
November 10, 2003


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

The invention provides cloned transgenic ungulates (e.g., bovines) in which prion protein activity is reduced by one or more genetically engineered mutations. Desirably, these transgenic bovines are also genetically modified to express xenogenous (e.g., human) antibodies. Because of their resistance to prion-related diseases such as bovine spongiform encephalopy (also known as mad cow disease), these bovines are a safer source of human antibodies for pharmaceutical uses and a safer source of agricultural products.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention features the design of knockout vectors with which very high frequency hemizygous and homozygous targeted integration (so-called homologous recombination) can be accomplished at the prion locus in ungulates (e.g., bovines) in donor cells such as fetal somatic fibroblasts. The invention also features methods for producing live calves having a hemizygous or homozygous mutation at the prion locus using genetically modified donor cells in any of the nuclear transfer methods described herein. These cattle are useful for producing pharmaceutical and agricultural products, such as therapeutic human antibodies for human use.

The method of the present invention is achieved using several technologies, such as (i) prion gene knockout cells described herein, (ii) mammalian cloning methods such as nuclear transfer or chromatin transfer described in PCT Publication No. WO02/051997, and (iii) introduction of a human artificial chromosome (HAC) such as delta HAC into an ungulate (PCT Publication No. WO02/70648; Kuroiwa et al., Nature Biotechnol. 20:889-894, 2002). The prion knockout ungulate cell is used as source of donor genetic material in a mammalian cloning method, resulting in prion knockout (hemi or homo) offspring.

Prion knockout ungulates may also have other useful features, such as production of human antibody. Such ungulates can be generated using a combination of the above technologies. For example, a prion knockout and human antibody-producing bovine can be generated by crossbreeding of a prion knockout ungulate and a human antibody-producing ungulate as described in PC Publication No. WO02/70648. Sequential manipulation of fetal fibroblast cells can also be used to generate such an ungulate, with or without breeding ungulates. Sequential manipulation of bovine fetal fibroblasts includes repeating the following steps: (i) genetic manipulation of an ungulate (e.g., a bovine) fibroblast cell, (ii) mammalian cloning using this cell, (iii) generation of a fetus, and (iv) isolation of a genetically-modified fetal fibroblast cell. For example, a prion knockout fibroblast cell can be sequentially manipulated to retain a HAC and to inactivate endogenous Ig genes.

The resulting monoclonal or polyclonal xenogenous antibodies have a variety of uses; for example, they may be used as ingredients in prophylactic or therapeutic compositions for infection of pathogenic microorganisms such as bacteria or viruses.

Transgenic Ungulates and Ungulate Cells

In one aspect, the invention provides an ungulate (e.g., bovine) or ungulate cell (e.g., bovine cell) having a non-naturally occurring mutation (e.g., a mutation after the initial ATC codon, such as a mutation that is within 10, 20, 50, or 100 nucleotides of this codon) in one or both alleles of an endogenous prion nucleic acid. Preferably, the mutation reduces or substantially eliminates the expression of functional prion protein. In preferred embodiments, expression of functional or total prion protein is decreased by at least 10, 20, 40, 60, 80, 90, 95, or 100%. The mutation may be hemizygous or homozygous. In some embodiments, the mutation includes an insertion of a positive selection marker (e.g., an antibiotic resistance gene) into the prion nucleic acid. Preferably, the positive selection marker is operably linked to a xenogenous promoter. For ungulates or ungulate cells with an antibiotic resistance gene inserted into both alleles of a prion nucleic acid, each allele may contain the same or a different antibiotic resistance gene. In a preferred embodiment, a negative selection marker (e.g., DT-A or Tk) is operably linked to a xenogenous promoter and is present in a vector used to mutate an endogenous prion allele. The mutation may or may not include the deletion of one or more nucleotides (e.g., contiguous nucleotides) in the prion nucleic acid.

In preferred embodiments of the above aspect, the ungulate (e.g., bovine) or ungulate cell (e.g., bovine cell) has one or more transgenes and expresses an mRNA or protein (e.g., antibody) encoded by the transgene(s). Preferred ungulates contain naturally arranged segments of human chromosomes (e.g., human chromosomal fragments) or artificial chromosomes that comprise artificially engineered human chromosome fragments (i.e., the fragments may be rearranged relative to the human genome). In some embodiments, the xenogenous nucleic acid is contained within a chromosome fragment. The nucleic acid may be integrated into a chromosome of the ungulate or maintained in the ungulate cell independently from the host chromosome. In various embodiments, the nucleic acid is contained in a chromosome fragment, such as a .DELTA.HAC or a .DELTA..DELTA.HAC. In other embodiments, the xenogenous antibody is an antibody from another genus, such as a human antibody.

Preferred ungulates and ungulate cells have one or more nucleic acids having a xenogenous antibody gene locus (e.g., a nucleic acid encoding all or part of a xenogenous immunoglobulin (Ig) gene that undergoes rearrangement and expresses at least one xenogenous Ig molecule) in one or more B-cells. Preferably, the nucleic acid has unrearranged antibody light chain nucleic acid segments in which all of the nucleic acid segments encoding a V gene segment are separated from all of the nucleic acid segments encoding a J gene segment by one or more nucleotides. Other preferred nucleic acids have unrearranged antibody heavy chain nucleic acid segments in which either (i) all of the nucleic acid segments encoding a V gene segment are separated from all of the nucleic acid segments encoding a D gene segment by one or more nucleotides and/or (ii) all of the nucleic acid segments encoding a D gene segment are separated from all of the nucleic acid segments encoding a J gene segment by one or more nucleotides. Other preferred ungulates have one or more nucleic acids encoding all or part of a rearranged xenogenous immunoglobulin (Ig) gene that expresses at least one xenogenous Ig molecule.

In other preferred embodiments, the light chain and/or heavy chain of the xenogenous antibodies is encoded by a human nucleic acid. In preferred embodiments, the heavy chain is any class of heavy chain, such as .mu., .gamma., .delta., .epsilon., or .alpha., and the light chain is a lambda or kappa light chain. In other preferred embodiments, the nucleic acid encoding the xenogenous immunoglobulin chain or antibody is in its unrearranged form. In other preferred embodiments, more than one class of xenogenous antibody is produced by the ungulate. In various embodiments, more than one different xenogenous Ig or antibody is produced by the ungulate. The xenogenous antibody may be a polyclonal or monoclonal antibody.

In various embodiments of the above aspect, the ungulate (e.g., bovine) or ungulate cell (e.g., bovine cell) has a mutation that reduces the expression of an endogenous antibody. Preferably, the mutation reduces the expression of functional IgM heavy chain or substantially eliminates the expression of functional IgM heavy chain. In other preferred embodiments, the mutation reduces the expression of functional Ig light chain or substantially eliminates the expression of functional Ig light chain. In yet other preferred embodiments, the mutation reduces the expression of functional IgM heavy chain and functional Ig light chain, or the mutation substantially eliminates the expression of functional IgM heavy chain and functional Ig light chain. Preferably, the ungulate also has a mutation in one or both alleles of an endogenous nucleic acid encoding alpha-(1,3)-galactosyltransferase and/or J chain. In other preferred embodiments, the ungulate has a nucleic acid encoding an exogenous J chain, such as a human J chain. Preferably, the mutation reduces or eliminates the expression of the endogenous alpha-(1,3)-galactosyltransferase enzyme, galactosyl(.alpha.1,3)galactose epitope, and/or J chain. Preferably, the ungulate produces human IgA or IgM molecules containing human J chain. Preferred ungulate cells (e.g., bovine cells) include, somatic cells, such as fetal fibroblasts or B-cells.

The invention also features hybridomas that produce xenogenous (e.g., human) antibodies. In one such aspect, the invention provides a hybridoma formed from the fusion of a B-cell of the invention with a myeloma cell.

Preferably, the antibody is reactive with an antigen of interest.

Methods for Producing Transgenic Ungulate Cells

The invention also features methods for generating ungulate cells (e.g., bovine cells) with a mutation in one or both alleles of a prion nucleic acid. These cells may be used as donor cells for the generation of transgenic ungulates (e.g., bovine prion knockout ungulates). Preferably, the mutation leads to a decrease in the amount of functional prion protein and/or a decreased incidence of prion related infections or diseases, such as BSE.

Accordingly, in one such aspect the invention features a method for producing a transgenic ungulate (e.g., bovine). This method involves introducing a first prion gene-targeting vector into an ungulate cell under conditions that allow homologous recombination between the first vector and a first allele of an endogenous prion nucleic acid in the cell, thereby introducing a hemizygous mutation in the cell. Preferably, the first vector includes a first region of homology having substantial sequence identity to a first region of an endogenous prion nucleic acid of the cell, a positive selection marker, and a second region of homology having substantial sequence identity to a second region of the prion nucleic acid. In a preferred embodiment, one region of homology is at least 1, 2, 3, 4, 5, 6, or 8 kilobases longer than the other region of homology. Preferably, the method also includes re-introducing the first vector into the cell under conditions that allow homologous recombination between the first vector and a second allele of an endogenous prion nucleic acid in the cell, thereby introducing a homozygous mutation in the cell. In other embodiments, the method also includes introducing a second prion gene targeting vector that has a different antibiotic resistance gene than the first vector into the cell under conditions that allow homologous recombination between the second vector and a second allele of an endogenous prion nucleic acid in the cell, thereby introducing a homozygous mutation in the cell. Preferably, the first and/or second vector is introduced into the cell in the presence of 1 mM of spermidine. Preferred cells include bovine fetal fibroblasts.

In various embodiments of the invention, the nucleic acid used to mutate an endogenous prion nucleic acid (e.g., a knockout cassette which includes a promoter operably linked to a nucleic acid encoding a selectable marker and operably linked to a nucleic acid having substantial sequence identity to a prion nucleic acid) is not contained in a viral vector, such as an adenoviral vector or an adeno-associated viral vector. For example, the nucleic acid may be contained in a plasmid or artificial chromosome that is inserted into an ungulate cell, using a standard method such as transfection or lipofection that does not involve viral infection of the cell. In yet another embodiment, the nucleic acid used to mutate an endogenous prion nucleic acid (e.g., a knockout cassette which includes a promoter operably linked to a nucleic acid encoding a selectable marker and operably linked to a nucleic acid having substantial sequence identity to a prion nucleic acid) is contained in a viral vector, such as an adenoviral vector or an adeno-associated viral vector. According to this embodiment, a virus containing the viral vector is used to infect an ungulate cell, resulting in the insertion of a portion or the entire viral vector into the ungulate cell.

Methods for Producing Transgenic Ungulates

The invention also provides methods for producing transgenic ungulates with one or more mutations in endogenous prion nucleic acids. One such method involves inserting a cell of any of the above aspects of the invention, a chromatin mass from the cell, or a nucleus from the cell into an oocyte. The cell has a first mutation in an endogenous prion nucleic acid. The oocyte or an embryo formed from the oocyte is transferred into the uterus of a host ungulate under conditions that allow the oocyte or the embryo to develop into a fetus. Preferably, the fetus develops into a viable offspring.

In preferred embodiments, the first mutation is introduced into the cell by inserting a nucleic acid comprising a cassette that includes a promoter operably linked to a nucleic acid encoding a selectable marker and operably linked to one or more nucleic acids having substantial sequence identity to an endogenous prion nucleic acid, whereby the cassette is integrated into one endogenous allele of a prion nucleic acid. In other preferred embodiments, the mutation is introduced in the cell by inserting into the cell a nucleic acid comprising a first cassette that includes a first promoter operably linked to a nucleic acid encoding a first selectable marker and operably linked to a first nucleic acid having substantial sequence identity to a endogenous prion nucleic acid, whereby the first cassette is integrated into a first endogenous allele of a prion nucleic acid producing a first transgenic cell. Into the first transgenic cell is inserted a nucleic acid that includes a second cassette that includes a second promoter operably linked to a nucleic acid encoding a second selectable marker and operably linked to a second nucleic acid having substantial sequence identity to a prion nucleic acid. The second selectable marker differs from the first selectable marker, and the second cassette is integrated into a second endogenous allele of a prion nucleic acid producing a second transgenic cell.

In still other preferred embodiments, a cell is isolated from the embryo, the fetus, or an offspring produced from the fetus, and another mutation is introduced into a prion nucleic acid or another nucleic acid (e.g., an antibody heavy or light chain nucleic acid) of the cell. A second round of nuclear transfer is then performed using the resulting cell, a chromatin mass from the cell, or a nucleus from the cell to produce a transgenic ungulate with two or more mutations. The mutations are in the same or different alleles of a gene or are in different genes. The cell used in the first or optional second round of nuclear transfer encodes a xenogenous antibody. In particular embodiments, the cell includes one or more nucleic acids encoding all or part of a xenogenous Ig gene that is capable of undergoing rearrangement and expressing one or more xenogenous Ig molecules in B-cells. In preferred embodiments, the cell that is mutated is a fibroblast (e.g., a fetal fibroblast). Preferably, the endogenous gene that is mutated is operably linked to an endogenous promoter that is not active in a fibroblast. In other preferred embodiments, the endogenous promoter operably linked to the endogenous gene that is mutated is less than 80, 70, 60, 50, 40, 30, 20, 10% as active as an endogenous promoter operably linked to a endogenous housekeeping gene such as GAPDH. Promoter activity may be measured using any standard assay, such as assays that measure the level of mRNA or protein encoded by the gene (see, for example, Ausubel et al. Current Protocols in Molecular Biology, volume 2, p. 11.13.1-11.13.3, John Wiley & Sons, 1995). This method for generating a transgenic ungulate has the advantage of allowing a gene that is not expressed in the donor cell (i.e., the cell that is the source of the genetic material used for nuclear transfer) to be mutated.

Preferably, the cell used in the production of the transgenic ungulate has a mutation in one or both alleles of an endogenous nucleic acid encoding alpha-(1,3)-galactosyltransferase and/or J chain. In other preferred embodiments, the cell has a nucleic acid encoding an exogenous J chain, such as a human J chain, or a nucleic acid encoding a xenogenous antibody. Preferably, the xenogenous nucleic acid encodes all or part of a xenogenous Ig gene, and the gene is capable of undergoing rearrangement and expressing more than one xenogenous Ig molecule in B-cells. In other preferred embodiments, the antibody is a polyclonal antibody. In yet other preferred embodiments, the immunogloblulin chain or antibody is expressed in serum and/or milk.

We have previously disclosed a variety of improved methods for cloning mammals (e.g., ungulates such as bovines) that may be used to clone mammals with one or more mutations in a prion nucleic acid (see, e.g., U.S. Patent Publication No. 2002-0046722 A1 and PCT Publication No. WO02/051997). In some of these methods, a permeabilized cell is incubated with a reprogramming media (e.g., a cell extract) to allow the addition or removal of factors from the cell, and then the plasma membrane of the permeabilized cell is resealed to enclose the desired factors and restore the membrane integrity of the cell. Some of these methods also involve the condensation of a donor nucleus (e.g., an isolated nucleus or a nucleus within a donor cell) into a chromatin mass to allow the release of nuclear components such as transcription factors that may promote the transcription of genes that are undesirable for the development of the nuclear transplant embryo into a viable offspring. If desired, the steps of any of these methods may be repeated one or more times or different reprogramming methods may be performed sequentially to increase the extent of reprogramming, resulting in greater viability of the cloned fetuses.

One method for generating a transgenic ungulate (e.g., bovine) involves incubating a permeabilized cell of any the above aspects of the invention (e.g., a cell that has one or more mutations in an endogenous prion nucleic acid) with a reprogramming media (e.g., a cell extract) under conditions that allow the removal of a factor (e.g., a nuclear or cytoplasmic component such as a transcription factor) from a nucleus, chromatin mass, or chromosome of the permeabilized cell or the addition of a factor to the nucleus, chromatin mass, or chromosome, thereby forming a reprogrammed cell. The reprogrammed cell is inserted into an enucleated oocyte, and the resulting oocyte or an embryo formed from the oocyte is transferred into the uterus of a host ungulate under conditions that allow the oocyte or embryo to develop into a fetus. In preferred embodiments, the permeabilized cell is contacted with one or more of the following under conditions that allow formation of a chromatin mass: a mitotic extract in the presence or absence of an anti-NuMA antibody, a detergent and/or salt solution, or a protein kinase solution. In yet another preferred embodiment, the permeabilized cell is incubated with an interphase reprogramming media (e.g., an interphase cell extract). In still another preferred embodiment, the nucleus in the permeabilized cell remains membrane-bounded, and the chromosomes in the nucleus do not condense during incubation with this interphase reprogramming media. In certain embodiments, incubating the permeabilized cell in the reprogramming media does not cause DNA replication or only causes DNA replication in less than 50, 40, 30, 20, 10, or 5% of the cells. In other embodiments, incubating the permeabilized cell in the reprogramming media causes DNA replication in at least 60, 70, 80, 90, 95, or 100% of the cells. In various embodiments, the permeabilized cell is formed by incubating an intact cell with a protease such as trypsin, a detergent, such as digitonin, or a bacterial toxin, such as Streptolysin O. In a preferred embodiment, the reprogrammed cell is not incubated under conditions that allow the membrane of the reprogrammed cell to reseal prior to insertion into the oocyte.

In yet another embodiment, the reprogrammed cell is incubated under conditions that allow the membrane of the reprogrammed cell to reseal prior to insertion into the oocyte. In other preferred embodiments, the reconstituted oocyte or the resulting embryo expresses lamin A, lamin C, or NuMA protein at a level that is less than 5-fold greater than the corresponding level expressed by a control oocyte or a control embryo with the same number of cells and from the same species.

In another aspect, the invention provides another method for generating a transgenic ungulate (e.g., bovine). This method involves (a) incubating a donor nucleus from a cell of the invention (e.g., a nucleus that has one or more mutations in an endogenous prion nucleic acid) under conditions that allow formation of a chromatin mass without causing DNA replication, (b) inserting the chromatin mass into an enucleated oocyte, thereby forming a nuclear transfer oocyte and (c) transferring the nuclear transfer oocyte or an embryo formed from the nuclear transfer oocyte into the uterus of a host ungulate under conditions that allow the nuclear transfer oocyte or embryo to develop into a fetus. In a preferred embodiment, the donor nucleus is incubated with a reprogramming media (e.g., a cell extract) under conditions that allow nuclear or cytoplasmic components such as transcription factors, repressor proteins, or chromatin remodeling proteins to be added to, or removed from, the nucleus or resulting chromatin mass. Preferably, the donor nucleus is contacted with one or more of the following under conditions that allow formation of a chromatin mass: a mitotic extract in the presence or absence of an anti-NuMA antibody, a detergent and/or salt solution, or a protein kinase solution. In other preferred embodiments, the reconstituted oocyte or the resulting embryo expresses lamin A, lamin C, or NuMA protein at a level that is less than 5-fold greater than the corresponding level expressed by a control oocyte or a control embryo with the same number of cells and from the same species. Preferably, the nucleus has fewer than four sets of homologous chromosomes (i.e., has fewer than two pairs of complete chromatids).

Preferred Methods for Generating Chimeric Ungulates

Other preferred ungulates are chimeric ungulates produced using cells from two or more embryos. For example, cells from a nuclear transfer embryo (e.g., an embryo formed by inserting a cell, nucleus, or chromatin mass into an enucleated oocyte) can be combined with cells from an in vitro fertilized, naturally-occurring, or parthenogenetically-activated embryo. Preferably, the majority of the cells and their progeny from the nuclear transfer embryo are incorporated into fetal tissue of the resulting chimeric embryo. At least some of the cells and their progeny from the second embryo are preferably incorporated into placental tissue and promote the viability of the resulting chimeric embryo. In preferred embodiments, the nuclear transfer embryo has a mutation in an endogenous prion nucleic acid and has a nucleic acid encoding a xenogenous antibody.

Accordingly, in one such aspect, the invention provides a method of producing a transgenic ungulate by inserting a cell, nucleus, or chromatin mass of the invention (e.g., a cell, nucleus, or chromatin mass having a mutation in an endogenous prion nucleic acid and optionally having one or more nucleic acids encoding a xenogenous antibody) into an oocyte, thereby forming a first embryo. One or more cells from the first embryo are contacted with one or more cells from a second embryo, thereby forming a third embryo. The second embryo is an in vitro fertilized embryo, naturally-occurring embryo, or parthenogenetically-activated embryo. The third embryo is transferred into the uterus of a host ungulate under conditions that allow the third embryo to develop into a fetus.

In another related aspect, the invention provides yet another method of generating a transgenic ungulate (e.g., a bovine). This method involves incubating a permeabilized cell of the invention (e.g., a cell having a mutation in an endogenous prion nucleic acid and optionally having one or more nucleic acids encoding a xenogenous antibody) in a reprogramming media (e.g., cell extract) under conditions that allow the removal of a factor from a nucleus, chromatin mass, or chromosome of the permeabilized cell or the addition of a factor from the reprogramming media to the nucleus, chromatin mass, or chromosome, thereby forming a reprogrammed cell. The reprogrammed cell is inserted into an enucleated oocyte, thereby forming a first embryo. One or more cells from the first embryo are contacted with one or more cells from an in vitro fertilized, naturally-occurring, or parthenogenetically-activated second embryo, forming a third embryo. The third embryo is transferred into the uterus of a host ungulate under conditions that allow the third embryo to develop into a fetus.

In a preferred embodiment, the permeabilized cell is incubated with a reprogramming media (e.g., a cell extract) under conditions that allow nuclear or cytoplasmic components such as transcription factors to be added to, or removed from, the nucleus or resulting chromatin mass. In other preferred embodiments, the permeabilized cell is contacted with one or more of the following under conditions that allow formation of a chromatin mass: a mitotic extract in the presence or absence of an anti-NuMA antibody, a detergent and/or salt solution, or a protein kinase solution. In yet another preferred embodiment, the permeabilized cell is incubated with an interphase reprogramming media (e.g., an interphase cell extract). In still another preferred embodiment, the nucleus in the permeabilized cell remains membrane-bounded, and the chromosomes in the nucleus do not condense during incubation with this interphase reprogramming media. In some embodiments, incubating the permeabilized cell in the reprogramming media does not cause DNA replication or only causes DNA replication in less than 50, 40, 30, 20, 10, or 5% of the cells. In other embodiments, incubating the permeabilized cell in the reprogramming media causes DNA replication in at least 60, 70, 80, 90, 95, or 100% of the cells. In various embodiments, the permeabilized cell is formed by incubating an intact cell with a protease such as trypsin, a detergent, such as digitonin, or a bacterial toxin, such as Streptolysin O. In a preferred embodiment, the reprogrammed cell is not incubated under conditions that allow the membrane of the reprogrammed cell to reseal prior to insertion into the oocyte. In yet another embodiment, the reprogrammed cell is incubated under conditions that allow the membrane of the reprogrammed cell to reseal prior to insertion into the oocyte.

In still other embodiments, the ungulate is generated by contacting a donor nucleus from a cell of the invention (e.g., a nucleus having a mutation in an endogenous prion nucleic acid and optionally having one or more nucleic acids encoding a xenogenous antibody) with a reprogramming media (e.g., cell extract) under conditions that allow formation of a chromatin mass, and inserting the chromatin mass into an enucleated oocyte, thereby forming a first embryo. One or more cells from the first embryo are contacted with one or more cells from an in vitro fertilized, naturally-occurring, or parthenogenetically activated second embryo, forming a third embryo. The third embryo is transferred into the uterus of a host ungulate under conditions that allow the third embryo to develop into a fetus. In a preferred embodiment, the chromatin mass is formed by contacting a donor nucleus that has less than four sets of homologous chromosomes with a reprogramming media under conditions that allow formation of a chromatin mass without causing DNA replication. Preferably, the donor nucleus is contacted with one or more of the following under conditions that allow formation of a chromatin mass: a mitotic extract in the presence or absence of an anti-NuMA antibody, a detergent and/or salt solution, or a protein kinase solution.

In preferred embodiments of any of the above aspects involving ungulates produced using cells from two embryos, at least one of the first embryo and the second embryo is a compaction embryo. In another embodiment, the first embryo and the second embryo are at different cell-stages. The first embryo and the donor cell used to produce the second embryo can be from the same species or from different genuses or species. Preferably, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% cells in the trophectoderm or placental tissue of the fetus are derived from the second embryo, or at least 30, 40, 50, 60, 70, 80, 90, 95, or 100% cells in the inner cell mass or fetal tissue of the fetus are derived from the first embryo. In other preferred embodiments, the first embryo or the third embryo expresses lamin A, lamin C, or NuMA protein at a level that is less than 5-fold greater than the corresponding level expressed by a control embryo with the same number of cells and from the same species.

In other preferred embodiments of any of the above aspects involving ungulates produced using cells from two embryos, part or all of the zona pellucida of the first embryo or second embryo is removed before the cells from each embryo are contacted. In one embodiment, the cells from the first and second embryos are contacted by being placed adjacent to each other in solution or on a solid support. In another embodiment, standard techniques are used to inject cells from the first embryo into the second embryo. The cells can be injected into any region of the second embryo, such as the periphery of the embryo between the zona pellucida and the embryo itself. Naturally occurring embryos include embryos that are surgically or nonsurgically removed from a pregnant ungulate (e.g., a bovine) using standard methods. In vitro fertilized embryos include intra-cytoplasmic sperm injection embryos generated using standard methods. It is also contemplated that cells from more than two embryos (e.g., cells from three, four, five, six, or more embryos) can be combined to form a chimeric embryo for generation of a cloned ungulate.
 

Claim 1 of 14 Claims

1. A bovine whose genome comprises a non-naturally occurring mutation in one or both alleles of an endogenous prion nucleic acid, wherein said mutation comprises a transcriptional termination sequence, and wherein said bovine exhibits reduced functional prion production.

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