The invention provides for transgenic donor animals (e.g., pigs) whose cells, tissues and organs have a better long-term survival when transplanted into a human patient. The transgenic donor animal carries one or more human transgenes which is expressed only when the endogenous gene of the donor animal is knocked out shortly before a graft is harvested for transplantation. This “genetic switch” allows the donor animal to remain healthy during the majority of its lifetime, while still allowing expression of the human transgene for optimal transplant tolerance in a human recipient. The transgene may encode a cytokine receptor, an adhesion molecule, or a complement regulatory protein.

The present invention provides transgenic animals (e.g., transgenic porcine animals), organs, tissues, and cells derived from the transgenic animals that are particularly useful for xenotransplantation therapies. In particular, the present invention provides transgenic porcine animals, as well as organs, tissues and cells derived from the transgenic porcine animals, which lack any expression of a functional alpha 1,3 galactosyltransferase (GTKO) gene and comprise at least six transgenes under the control of at least three promoters within a single multi-gene expression vector, and further comprise at least four additional genetic modifications. Also provided are methods of making the transgenic animals (e.g., transgenic porcine animals), and methods of using the transgenic animals, organs, tissues, and cells derived from the transgenic animals for xenotransplantation therapies and treating a disease or condition.

Provided is a method for developing a secondary organ by using a non-human animal in which organ formation is inhibited, for the purpose of establishing a process for producing a functional cell such as a β cell within the body of an animal such as a pig, the method including the step of raising a newborn or a fetus of the non-human animal in which organ formation is inhibited by complementing at least a part of the function of the organ whose formation is inhibited.

Provided is an SgRNA combination, comprising an SgRNA specifically targeting the GGTA1 gene, an SgRNA specifically targeting the CMAH gene and an SgRNA specifically targeting the β4GalNT2 gene. Also provided is a CRISPR/Cas9 vector combination, comprising a GGTA1-CRISPR/Cas9 vector, a CMAH-CRISPR/Cas9 vector and a β4GalNT2-CRISPR/Cas9 vector. Also provided is an applicaton of the CRISPR/Cas9 vector combination in knocking out the GGTA1 gene, the CMAH gene and the β4GalNT2 gene. The knockout rates of the three genes with the specifically targeted SgRNA sequences are respectively 56%, 63%, and 41%. A three genes knockoutpig can be obtained, wherein the three genes related to immune rejectionare knocked out, and heart valves of said pig can be acquired.

The present invention is a porcine animal, tissue, organ, cells and cell lines, which lack any expression of functional alpha 1,3 galactosyltransferase (alpha1,3GT). These animals, tissues, organs and cells can be used in xenotransplantation and for other medical purposes.

Provided is a method for developing a secondary organ by using a non-human animal in which organ formation is inhibited, for the purpose of establishing a process for producing a functional cell such as a β cell within the body of an animal such as a pig, the method including the step of raising a newborn or a fetus of the non-human animal in which organ formation is inhibited by complementing at least a part of the function of the organ whose formation is inhibited.

A genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ comprising live cells that vascularize after xenotransplantation, wherein the genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ has been obtained from a non-wild type, biologically engineered porcine comprising a nuclear genome that has been reprogrammed to replace a plurality of nucleotides in a plurality of exon regions of a major histocompatibility complex of a wild-type porcine with a plurality of synthesized nucleotides from a human captured reference sequence, wherein the nuclear genome has wild-type porcine intron regions from a wild-type porcine and has been reprogrammed at exon regions.

Described herein is a method for producing a chimeric non-human animal expressing a human ETV2 gene comprising: a) generating an ETV2 null non-human animal cell, wherein both copies of the non-human ETV2 gene carry a mutation that prevents production of functional ETV2 protein in said non-human animal; b) creating an ETV2 null non-human blastocyst by somatic cell nuclear transfer comprising fusing a nucleus from said ETV2 null non-human animal cell of a) into an enucleated non-human oocyte and activating said oocyte to divide so as to form an ETV2 null non-human blastocyst; c) introducing human stem cells into the ETV2 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 ETV2.

A biological system for generating and preserving a repository of personalized, humanized transplantable cells, tissues, and organs for transplantation, wherein the biological system is biologically active and metabolically active, the biological system having genetically reprogrammed cells, tissues, and organs in a non-human animal for transplantation into a human recipient, wherein the non-human animal does not present one or more surface glycan epitopes and specific sequences from the wild-type swine's SLA is replaced with a synthetic nucleotides based on a human captured reference sequence from a human recipient's HLA.

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 at a high percentage is sustained for a long period of time. 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, When a medium to large mammal 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.