The present invention provides livestock animals and methods to create recipient animals for spermatogonial stem cell transplantation through modulation of the NANOS gene. In one embodiment genome editing issued to create animals with insertions or deletions (indels) that inactivate or otherwise modulate NANOS gene activity so that resulting males lack functional germ cells yet retain functional somatic cells, and females are fertile. These males can then be transplanted with donor spermatogonial stem cells and used for breeding.

Described herein is a method for producing a chimeric non-human animal expressing a human a MYF5, MYOD, MRF4 gene or a combination thereof gene comprising: a) generating an MYF5, MYOD, MRF4 or combination thereof null non-human animal cell, wherein both copies of the non-human MYF5, MYOD, MRF4 gene or combination thereof carry a mutation that prevents production of functional MYF5, MYOD, MRF4 protein or combination thereof in said non-human animal; b) creating a MYF5, MYOD, MRF4 or combination thereof null non-human blastocyst by somatic cell nuclear transfer comprising fusing a nucleus from said MYF5, MYOD, MRF4 or combination thereof null non-human animal cell of a) into an enucleated non-human oocyte and activating said oocyte to divide so as to form an MYF5, MYOD, MRF4 or combination thereof null non-human blastocyst; c) introducing human stem cells into the MYF5, MYOD, MRF4 or combination null non-human blastocyst of b); and d) implanting said blastocyst from c) into a pseudopregnant surrogate non-human animal to generate a chimeric non-human animal expressing human MYF5, MYOD, MRF4 or combination thereof.

Genetically modified animals suitable for use in xenotransplantation and methods of producing such animals suitable for use in xenotransplantation are provided. These animals and methods are particularly suited to human xenotransplantation. Specifically, the present application describes the production of genetically modified pigs lacking expression of glycoprotein alpha-galactosyltransferase 1 (GGTA1), cytidine monophospho-n-acetylneuraminic acid hydroxylase (CMAH), and beta-1,4-n-acetyl-galactosaminyltransferase 2 (B4GALNT2); and expressing human transgenes including human thrombomodulin (hTBM), human membrane cofactor protein (hCD46), and human decay accelerating factor for complement (hCD55). Preferably, the genetically modified pigs are Yucatan miniature pigs, and organs, tissues and cells derived therefrom are also provided. Method of inducing immune tolerance prior to the xenotransplantation by transferring the organ recipient's cells and factors into fetal pig donors and back to the organ recipient after the birth and development of the donor animal are also provided.

The present invention is directed to transgenic animals (e.g., transgenic porcine animals) comprising multiple genetic modifications that advantageously render these animals suitable donors for xenotransplanation. The present invention extends to organs, organ fragments, tissues and cells derived from these animals and their therapeutic use. The present invention further extends to methods of making such animals. In certain embodiments, the transgenic animals (e.g., transgenic porcine animals) lack expression of alpha gal and incorporate and express at least four transgenes under the control of at least two promoters.

The present application discloses a method for generating human tissues in a non-human animal comprising: (i) generating human nave state stem cells and injecting them into a blastocyst or embryo of a non-human animal such that a chimeric animal is generated; (ii) harvesting human tissues, organs, cells or factors secreted by or made in the human tissues or cells from the chimeric animal; and (iii) transplanting or administering the harvested material into a human resulting in generation of human tissues in a non-human animal.

The application provides methods of improving a rejection related symptom, reducing premature separation and methods of producing a compound of interest with an altered epitope profile are provided. Transgenic pigs with a disrupted gene or genes, and porcine organs, tissues, and cells therefrom are provided.

Described herein is a method for producing a chimeric non-human animal expressing a human a MYF5, MYOD, MRF4 gene or a combination thereof gene comprising: a) generating an MYF5, MYOD, MRF4 or combination thereof null non-human animal cell, wherein both copies of the non-human MYF5, MYOD, MRF4 gene or combination thereof carry a mutation that prevents production of functional MYF5, MYOD, MRF4 protein or combination thereof in said non-human animal; b) creating a MYF5, MYOD, MRF4 or combination thereof null non-human blastocyst by somatic cell nuclear transfer comprising fusing a nucleus from said MYF5, MYOD, MRF4 or combination thereof null non-human animal cell of a) into an enucleated non-human oocyte and activating said oocyte to divide so as to form an MYF5, MYOD, MRF4 or combination thereof null non-human blastocyst; c) introducing human stem cells into the MYF5, MYOD, MRF4 or combination null non-human blastocyst of b); and d) implanting said blastocyst from c) into a pseudopregnant surrogate non-human animal to generate a chimeric non-human animal expressing human MYF5, MYOD, MRF4 or combination thereof.

Described herein is a method for producing a chimeric non-human animal expressing a human NKX2-5, HANDII, TBX5 gene or a combination thereof gene comprising: a) generating a NKX2-5, HANDII, TBX5 or combination thereof null non-human animal cell, wherein both copies of the non-human NKX2-5, HANDII, TBX5 gene or combination thereof carry a mutation that prevents production of functional NKX2-5, HANDII, TBX5 protein or combination thereof in said non-human animal; b) creating a NKX2-5, HANDII, TBX5 or combination thereof null non-human blastocyst by somatic cell nuclear transfer comprising fusing a nucleus from said NKX2-5, HANDII, TBX5 or combination thereof null non-human animal cell of a) into an enucleated non-human oocyte and activating said oocyte to divide so as to form an NKX2-5, HANDII, TBX5 or combination thereof null non-human blastocyst; c) introducing human stem cells into the NKX2-5, HANDII, TBX5 or combination null non-human blastocyst of b); and d) implanting said blastocyst from c) into a pseudopregnant surrogate non-human animal to generate a chimeric non-human animal expressing human NKX2-5, HANDII, TBX5 or combination thereof.

The present invention discloses a novel means capable of producing a blood chimeric animal in which a state of retaining blood cells originating in a heterologous animal, such as a human, at a high percentage is sustained for a long period of time, even in cases where a medium to large domestic animal is used as a recipient. The method for producing a non-human animal that retains blood cells originating in a heterologous animal, according to the present invention, comprises transplanting hematopoietic cells of a heterologous animal into a non-human animal, in which hematopoietic cells the function of a gene that acts on the hematopoietic system is modified. The gene that acts on the hematopoietic system is, for example, Lnk gene. According to the method of the present invention, when a medium to large mammal such as a pig is used as a recipient, the survival rate of hematopoietic cells originating in a heterologous animal is dramatically increased such that blood chimerism of 10% or more can be maintained even in a 16 month old animal.

A method of modulating some or all copies of a gene in a cell is provided including introducing into a cell one or more ribonucleic acid (RNA) sequences that comprise a portion that is complementary to all or a portion of each of the one or more target nucleic acid sequences, and a nucleic acid sequence that encodes a Cas protein and maintaining the cells under conditions in which the Cas protein is expressed and the Cas protein binds and modulates the one or more target nucleic acid sequences in the cell.