Genetically modified non-human animals and methods of use thereof
The invention relates generally to genetically modified non-human animals expressing human polypeptides and their methods of use.
1. A method for generating a genetically modified, immunodeficient mouse comprising in its genome a replacement of a mouse M-CSF gene with a nucleic acid encoding a human M-CSF polypeptide at a mouse M-CSF gene locus, a replacement of a mouse IL-3 gene with a nucleic acid encoding a human IL-3 polypeptide at a mouse IL-3 gene locus, a replacement of a mouse GM-CSF gene with a nucleic acid encoding a human GM-CSF polypeptide at a mouse GM-CSF gene locus, an insertion of a nucleic acid encoding a human SIRPA polypeptide, and a replacement of a mouse TPO gene with a nucleic acid encoding a human TPO polypeptide at a mouse TPO gene locus, wherein each of the nucleic acids encoding the human M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSF polypeptide, the human SIRPA polypeptide, and the human TPO polypeptide is operably linked to a promoter, and wherein the mouse expresses the human M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSF polypeptide, the human SIRPA polypeptide, and the human TPO polypeptide, the method comprising:
breeding a first mouse with a second mouse,
wherein the first mouse comprises in its genome a replacement of a mouse M-CSF gene with a nucleic acid encoding a human M-CSF polypeptide at a mouse M-CSF gene locus, a replacement of a mouse IL-3 gene with a nucleic acid encoding a human IL-3 polypeptide at a mouse IL-3 gene locus, a replacement of a mouse GM-CSF gene with a nucleic acid encoding a human GM-CSF polypeptide at a mouse GM-CSF gene locus, and a replacement of a mouse TPO gene with a nucleic acid encoding a human TPO polypeptide at a mouse TPO gene locus, and
the second mouse comprises in its genome an insertion of a nucleic acid encoding a human SIRPA polypeptide, a Rag-2 gene knock-out, and an IL2rg gene knock-out,
wherein each of the nucleic acids encoding the human M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSF polypeptide, the human SIRPA polypeptide, and the human TPO polypeptide is operably linked to a promoter, to generate the genetically modified, immunodeficient mouse.
2. The method of claim 1 , wherein the genetically modified, immunodeficient mouse comprises in its genome a recombination activating gene 2 (Rag-2) gene knock-out and an IL2 receptor gamma chain (IL2rg) gene knock-out.
3. The method of claim 1 , wherein the genetically modified, immunodeficient mouse has severe combined immune deficiency (SCID) or is a NOD Scid γ c −/− (NOG) mouse.
4. The method of claim 1 , wherein the first mouse has the genotype M-CSF h/h IL-3 h/h GM-CSF h/h TPO h/h .
5. The method of claim 1 , wherein the second mouse has the genotype hSIRPA tg RAG2 −/− γc −/−.
6. The method of claim 4 , wherein the second mouse has the genotype hSIRPA tg RAG2 −/− γc −/− .
7. The method of claim 1 , wherein the human SIRPA polypeptide is a biologically active fragment of a full-length human SIRPA polypeptide.
8. The method of claim 1 , wherein the human SIRPA polypeptide is a full-length human SIRPA polypeptide.
9. The method of claim 1 , wherein the nucleic acid encoding the human SIRPA polypeptide is operably linked to a human SIRPA promoter.
10. The method of claim 1 , wherein the nucleic acid encoding the human M-CSF polypeptide is operably linked to a mouse M-CSF promoter.
11. The method of claim 1 , wherein the nucleic acid encoding the human IL-3 polypeptide is operably linked to a mouse IL-3 promoter.
12. The method of claim 1 , wherein the nucleic acid encoding the human GM-CSF polypeptide is operably linked to a mouse GM-CSF promoter.
13. The method of claim 1 , wherein the nucleic acid encoding the human TPO polypeptide is operably linked to a mouse TPO promoter.
14. The method of claim 9 , wherein the nucleic acid encoding the human M-CSF polypeptide is operably linked to a mouse M-CSF promoter.
15. The method of claim 14 , wherein the nucleic acid encoding the human IL-3 polypeptide is operably linked to a mouse IL-3 promoter.
16. The method of claim 15 , wherein the nucleic acid encoding the human GM-CSF polypeptide is operably linked to a mouse GM-CSF promoter.
17. The method of claim 16 , wherein the nucleic acid encoding the human TPO polypeptide is operably linked to a mouse TPO promoter.
18. The method of claim 17 , wherein the genetically modified, immunodeficient mouse comprises in its genome a recombination activating gene 2 (Rag-2) gene knock-out and an IL2 receptor gamma chain (IL2rg) gene knock-out.