Humanized T cell co-receptor mice
The invention provides genetically modified non-human animals that express chimeric human/non-human T cell co-receptor polypeptides (e.g., CD4, CD8α, CD8β), as well as embryos, cells, and tissues comprising the same. Also provided are constructs for making said genetically modified animals and methods of making the same.
1. A genetically modified rodent cell comprising:
(i) a first nucleotide sequence encoding a chimeric human/rodent CD8α polypeptide, wherein the amino acid sequence of the human portion of the chimeric human/rodent CD8α polypeptide (a) is the amino acid sequence set forth in SEQ ID NO:59 and (b) is operably linked to transmembrane and cytoplasmic domains of a rodent CD8α polypeptide, and
(ii) a second nucleotide sequence encoding a chimeric human/rodent CD8β polypeptide, wherein the amino acid sequence of the human portion of the chimeric human/rodent CD8β polypeptide (a) comprises at least an immunoglobulin V-like domain of a human CD8β polypeptide and (b) is;
operably linked to transmembrane and cytoplasmic domains of a rodent CD8β polypeptide;
wherein the cell is an embryonic stem cell.
2. The genetically modified rodent cell of claim 1 , wherein the first nucleotide sequence and the second nucleotide sequence are present at an endogenous CD8 locus.
3. The genetically modified rodent cell of claim 1 , wherein the rodent cell is a rat cell.
4. The genetically modified rodent cell of claim 1 , wherein the rodent cell is a mouse cell.
5. The genetically modified rodent cell of claim 4 , wherein:
(i) the first nucleotide sequence encodes a chimeric human/mouse CD8α polypeptide, and
the amino acid sequence of the human portion of the chimeric human/mouse CD8α polypeptide set forth in SEQ ID NO:59 is operably linked to transmembrane and cytoplasmic domains of a mouse CD8α polypeptide, and
(ii) the second nucleotide sequence encodes a chimeric human/mouse CD8β polypeptide, and the human portion of the chimeric human/mouse CD8β polypeptide comprising the immunoglobulin V-like domain of the human CD8β polypeptide;
is operably linked to transmembrane and cytoplasmic domains of a mouse CD8β polypeptide.
6. The genetically modified rodent cell of claim 5 , wherein
(i) the first nucleotide sequence is present at an endogenous CD8α locus, and
(ii) the second nucleotide sequence is present at an endogenous CD8β locus.
7. The genetically modified rodent cell of claim 6 , wherein the amino acid sequence of the human portion of the chimeric human/mouse CD8β polypeptide is the amino acid sequence set forth in SEQ ID NO:58.
8. The genetically modified rodent cell of claim 7 , wherein (i) the chimeric human/mouse CD8α polypeptide comprises the amino acid sequence set forth in SEQ ID NO:54 and (ii) the chimeric human/mouse CD8β polypeptide comprises the amino acid sequence set forth in SEQ ID NO:53.
9. A rodent embryo developed from the rodent embryonic stem cell of claim 1 .
10. A method of modifying a mouse embryonic stem cell comprising replacing:
(i) at an endogenous CD8α locus, a nucleotide sequence encoding an endogenous CD8α polypeptide extracellular domain with a first orthologous nucleotide sequence encoding the human amino acid sequence set forth in SEQ ID NO:59, and
(ii) at an endogenous CD8β locus, a nucleotide sequence encoding an endogenous CD8β polypeptide extracellular domain and comprising at least exons 2-3 of an endogenous mouse CD8β gene with a second orthologous nucleotide sequence encoding a human CD8β polypeptide extracellular domain.
11. The method of claim 10 , wherein the amino acid sequence of the human CD8β polypeptide extracellular domain is the amino acid sequence set forth in SEQ ID NO:58.
12. The method of claim 10 , wherein the endogenous CD8α and CD8β loci respectively encode a chimeric human/mouse CD8α polypeptide that comprises the amino acid sequence set forth in SEQ ID NO:54 and a chimeric human/mouse CD8β polypeptide that comprises the amino acid sequence set forth in SEQ ID NO:53.
13. A genetically modified rodent cell comprising:
(i) a first nucleotide sequence encoding a chimeric human/rodent CD8α polypeptide, wherein the amino acid sequence of the human portion of the chimeric human/rodent CD8α polypeptide (a) is the amino acid sequence set forth in SEQ ID NO:59 and (b) is operably linked to transmembrane and cytoplasmic domains of a rodent CD8α polypeptide, and
(ii) a second nucleotide sequence encoding a chimeric human/rodent CD8β polypeptide, wherein the amino acid sequence of the human portion of the chimeric human/rodent CD8β polypeptide (a) comprises at least an immunoglobulin V-like domain of a human CD8β polypeptide and (b) is;
operably linked to a nucleic acid sequence encoding transmembrane and cytoplasmic domains of rodent CD8β polypeptide,
wherein the cell is a T cell that expresses a chimeric human/rodent CD8 protein comprising the chimeric human/rodent CD8α and the chimeric human/rodent CD8β polypeptides, and
wherein the chimeric human/rodent CD8 protein interacts with a human or humanized MHC I polypeptide.
14. The genetically modified rodent cell of claim 13 , wherein the T cell does not express a functional endogenous rodent CD8 co-receptor from its endogenous CD8 locus.
15. The genetically modified rodent cell of claim 13 , wherein the T cell does not express CD4.
16. The genetically modified rodent cell of claim 13 , wherein the first nucleotide sequence and the second nucleotide sequence are present at an endogenous CD8 locus.
17. The genetically modified rodent cell of claim 13 , wherein the rodent cell is a rat cell.
18. The genetically modified rodent cell of claim 13 , wherein the rodent cell is a mouse cell.
19. The genetically modified rodent cell of claim 18 , wherein:
(i) the first nucleotide sequence encodes a chimeric human/mouse CD8α polypeptide, and
the amino acid sequence of the human portion of the chimeric human/mouse CD8α polypeptide set forth in SEQ ID NO:59 is operably linked to transmembrane and cytoplasmic domains of a mouse CD8α polypeptide, and
(ii) the second nucleotide sequence encodes a chimeric human/mouse CD8β polypeptide, and the immunoglobulin V-like domain of the human CD8β polypeptide;
is operably linked to transmembrane and cytoplasmic domains of a mouse CD8β polypeptide.
20. The genetically modified rodent cell of claim 19 , wherein
(i) the first nucleotide sequence is present at an endogenous CD8α locus, and
(ii) the second nucleotide sequence is present at an endogenous CD8β locus.
21. The genetically modified rodent cell of claim 20 , wherein the amino acid sequence of the human portion of the chimeric human/mouse CD8β polypeptide is the amino acid sequence set forth in SEQ ID NO:58.
22. The genetically modified rodent cell of claim 21 , wherein the chimeric human/mouse CD8α polypeptide comprises the amino acid sequence set forth in SEQ ID NO:54 and the chimeric human/mouse CD8β polypeptide comprises the amino acid sequence set forth in SEQ ID NO:53.
23. A rodent tissue comprising the rodent T cell of claim 13 .
24. A chimeric human/rodent CD8α polypeptide that comprises a human extracellular domain having the amino acid sequence set forth in SEQ ID NO:59 operably linked to at least transmembrane and cytoplasmic domains of a rodent CD8α polypeptide.
25. The chimeric human/rodent CD8α polypeptide of claim 24 , wherein the rodent CD8α polypeptide is a rat CD8α polypeptide.
26. The chimeric human/rodent CD8α polypeptide of claim 24 , wherein the rodent CD8α polypeptide is a mouse CD8α polypeptide.
27. A chimeric human/rodent CD8 protein comprising a chimeric human/rodent CD8α polypeptide and a chimeric human/rodent CDβ polypeptide,
wherein the chimeric human/rodent CD8α polypeptide comprises a human extracellular domain having the amino acid sequence set forth in SEQ ID NO:59, operably linked to at least transmembrane and cytoplasmic domains of a rodent CD8α polypeptide, and
the chimeric human/rodent CDβ polypeptide comprises a human extracellular domain having the amino acid sequence set forth in SEQ ID NO:58, operably linked to at least transmembrane and cytoplasmic domains of a rodent CD8β polypeptide.
28. The chimeric human/rodent CD8 protein of claim 27 , wherein the rodent CD8β polypeptide is a rat CD8β polypeptide.
29. The chimeric human/rodent CD8 protein of claim 27 , wherein the rodent CD8β polypeptide is a mouse CD8β polypeptide.