Non-human animals having a humanized TSLP gene, a humanized TSLP receptor gene, and / or a humanized IL7RA gene

Abstract

Disclosed herein are rodents (such as, but not limited to, mice and rats) that have been genetically modified to include a humanized Tslp gene, a humanized Tslpr gene, a humanized Il7ra gene, or a combination thereof. Provided are compositions and methods for making such genetically modified rodents, as well as methods for using such genetically modified rodents as animal models for diseases, such as allergic diseases and cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 128,258, filed December 21, 2020, the entire contents of which are incorporated herein by reference.

[0002] Incorporation by reference of sequence listing A sequence listing in the form of a 192KB ASCII text file entitled 37301_10589WO01_SequenceListing, created on December 2, 2021 and submitted to the U.S. Patent and Trademark Office via EFS-Web, is incorporated herein by reference. [Background technology]

[0003] Thymic stromal lymphopoietin (TSLP) acts through a heterodimeric receptor composed of a TSLP-specific chain (termed TSLPR or Tslpr) and the IL7 receptor α chain and has been implicated in allergic diseases and certain cancers. Effective in vivo systems are desired to gain a better understanding of the pathogenesis of allergic diseases and cancer and for the development of therapeutic approaches. Summary of the Invention

[0004] In some embodiments, disclosed herein is a genetically modified rodent comprising in its genome a humanized Tslp gene, the humanized Tslp gene comprising a rodent Tslp nucleic acid sequence and a human TSLP nucleic acid sequence, and the humanized Tslp gene encodes a humanized Tslp polypeptide comprising a mature protein sequence substantially identical to the mature protein sequence of the human TSLP protein.

[0005] In some embodiments, the humanized Tslp polypeptide comprises a mature protein sequence identical to that of a human TSLP protein.

[0006] In some embodiments, the humanized Tslp protein comprises a signal peptide substantially identical to that of a rodent Tslp protein, hi some embodiments, the humanized Tslp protein comprises a signal peptide identical to that of a rodent Tslp protein, e.g., an endogenous rodent Tslp protein.

[0007] In some embodiments, the human TSLP nucleic acid sequence in the humanized Tslp gene encodes at least a substantial portion of the mature protein sequence of the human TSLP protein. In some embodiments, the human TSLP nucleic acid sequence encodes the mature protein sequence of the human TSLP protein, e.g., amino acids 29-159 of a human TSLP protein (e.g., the human TSLP protein set forth in SEQ ID NO:3). In some embodiments, the human TSLP nucleic acid sequence includes exon 1 from the codon encoding the first amino acid of the mature protein sequence of the human TSLP gene through the stop codon in exon 4.

[0008] In some embodiments, the rodent Tslp nucleic acid sequence in the humanized Tslp gene comprises an exon sequence of a rodent Tslp gene (e.g., an endogenous rodent Tslp gene) that encodes a rodent Tslp signal peptide. In some embodiments, the rodent is a mouse, and the rodent nucleic acid sequence in the humanized Tslp gene comprises exon 1, which encodes the signal peptide amino acids, and a 5' portion of exon 2 of the mouse Tslp gene. In some embodiments, the rodent Tslp nucleic acid sequence in the humanized Tslp gene also comprises the 3'UTR of a rodent Tslp gene (e.g., an endogenous rodent Tslp gene).

[0009] In some embodiments, the rodent is a mouse and the humanized Tslp gene comprises (i) exon 1, which encodes the signal peptide amino acid, and a 5' portion of exon 2 of the mouse Tslp gene, and (ii) exon 1 from the codon encoding the first amino acid of the mature protein sequence of the human TSLP gene, up to the stop codon in exon 4. In some embodiments, the humanized Tslp gene further comprises the 3' UTR of the mouse Tslp gene. In various embodiments, the mouse Tslp gene is an endogenous mouse Tslp gene.

[0010] In some embodiments, the humanized Tslp gene is operably linked to a rodent Tslp promoter, such as an endogenous rodent Tslp promoter.

[0011] In some embodiments, the humanized Tslp gene is located at a locus other than the endogenous rodent Tslp locus.In some embodiments, the humanized Tslp gene is located at the endogenous rodent Tslp locus.

[0012] In some embodiments, the humanized Tslp gene is located at the endogenous rodent Tslp locus, the humanized Tslp gene is formed as a result of the replacement of rodent Tslp genomic DNA at the endogenous rodent Tslp locus with human TSLP nucleic acid. In some embodiments, the humanized Tslp gene is formed as a result of the replacement of rodent genomic DNA comprising exon sequence encoding at least a substantial portion of the mature protein sequence of endogenous rodent Tslp protein with human TSLP nucleic acid encoding at least a substantial portion of the mature protein sequence of human TSLP protein. In some embodiments, the humanized Tslp gene is formed as a result of the replacement of rodent genomic DNA comprising exon sequence encoding at least a substantial portion of the mature protein sequence of endogenous rodent Tslp protein with human TSLP nucleic acid encoding at least a substantial portion of the mature protein sequence of human TSLP protein. In some embodiments, the rodent is a mouse, and the replaced mouse genomic DNA includes exon 2 from the codon encoding the first amino acid of the mature mouse Tslp protein of the endogenous mouse Tslp gene to a stop codon in exon 5, and the human genomic DNA includes exon 1 from the codon encoding the first amino acid of the mature human TSLP protein of the human TSLP gene to a stop codon in exon 4.

[0013] In some embodiments, the rodent is homozygous for the humanized Tslp gene. In some embodiments, the rodent is heterozygous for the humanized Tslp gene.

[0014] In some embodiments, the humanized Tslp polypeptide is expressed from a humanized Tslp gene in a rodent.

[0015] In some embodiments, the rodent further comprises a humanized Tslpr gene, a humanized Il7ra gene, or a combination thereof in its genome.

[0016] In some embodiments, the rodent is a mouse or a rat.

[0017] In some embodiments, disclosed herein is an isolated rodent cell or tissue comprising in its genome a humanized Tslp gene as described herein. In some embodiments, the rodent cell is a rodent embryonic stem cell. In some embodiments, the rodent cell is an egg or sperm. In some embodiments, the isolated rodent tissue or cell is a mouse tissue or cell, or a rat tissue or cell.

[0018] Disclosed herein, in some embodiments, is a rodent embryo comprising a rodent embryonic stem cell comprising a humanized Tslp gene as described herein.

[0019] In some embodiments, disclosed herein are methods of generating a genetically modified rodent. In some embodiments, the methods include modifying a rodent genome to contain a humanized Tslp gene, the humanized Tslp gene comprising a rodent Tslp nucleic acid sequence and a human TSLP nucleic acid sequence, and encoding a humanized Tslp polypeptide comprising a mature protein sequence substantially identical to the mature protein sequence of the human TSLP protein, and generating a rodent comprising the modified rodent genome.

[0020] In some embodiments, modifying the rodent genome comprises introducing a nucleic acid molecule comprising a human TSLP nucleic acid sequence into the genome of a rodent embryonic stem (ES) cell, obtaining a rodent ES cell in which the human TSLP nucleic acid sequence is integrated into the endogenous rodent Tslp locus to replace the rodent Tslp genomic DNA, thereby forming a humanized Tslp gene, and generating a rodent from the resulting rodent ES cell. In some embodiments, the human TSLP nucleic acid sequence encodes at least a substantial portion of the mature protein sequence of the human TSLP protein. In some embodiments, the nucleic acid molecule introduced into the ES cell further comprises 5' and 3' homology arms flanking the human TSLP nucleic acid sequence, the 5' and 3' homology arms being homologous to the nucleic acid sequence at the endogenous rodent locus flanking the rodent Tslp genomic DNA being replaced. In some embodiments, the humanized Tslp gene is operably linked to a rodent Tslp promoter, for example an endogenous rodent Tslp promoter at an endogenous rodent Tslp locus.

[0021] In some method embodiments, the rodent is a mouse or a rat.

[0022] In some embodiments, disclosed herein are targeting nucleic acid constructs comprising a human TSLP nucleic acid sequence flanked by 5' and 3' nucleotide sequences that are homologous to nucleotide sequences at the rodent Tslp locus that are integrated into a rodent Tslp gene at the endogenous rodent Tslp locus, where integration of the human TSLP nucleic acid sequence into the rodent Tslp gene results in replacement of the rodent Tslp genomic DNA with the human TSLP nucleic acid sequence, thereby forming a humanized Tslp gene, where the human TSLP nucleic acid sequence encodes at least a substantial portion of the mature protein sequence of the human TSLP protein. In some targeting nucleic acid embodiments, the rodent is a rat or a mouse.

[0023] In some embodiments, an in vitro method for generating a genetically modified rodent cell is disclosed herein, comprising introducing into a rodent cell a targeting vector comprising a human TSLP nucleic acid sequence encoding at least a substantial portion of the mature protein sequence of the human TSLP protein flanked by rodent homology arms that mediate integration of the human TSLP nucleotide sequence into the endogenous rodent Tslp locus, resulting in replacement of the rodent Tslp genomic DNA with the human TSLP nucleic acid sequence to form a humanized Tslp gene as described herein, thereby generating a genetically modified rodent cell. In some embodiments, the rodent cell is a mouse cell or a rat cell. In some embodiments, the rodent cell is a rodent ES cell, and the method generates a genetically modified rodent ES cell.

[0024] In some embodiments, disclosed herein is a genetically modified rodent comprising in its genome a humanized Tslpr gene, wherein the humanized Tslpr gene comprises a rodent Tslpr nucleic acid sequence and a human TSLPR nucleic acid sequence, and wherein the humanized Tslpr gene encodes a humanized Tslpr polypeptide comprising an extracellular domain substantially identical to the extracellular domain of the human TSLPR protein.

[0025] In some embodiments, the humanized Tslpr protein comprises a transmembrane cytoplasmic sequence that is substantially identical to the transmembrane cytoplasmic sequence of a rodent Tslpr protein (e.g., an endogenous rodent Tslpr protein). In some embodiments, the humanized Tslpr protein comprises a transmembrane cytoplasmic sequence that is substantially identical to the transmembrane cytoplasmic sequence of a rodent Tslpr protein (e.g., an endogenous rodent Tslpr protein).

[0026] In some embodiments, the humanized Tslpr protein comprises a signal peptide substantially identical to that of a rodent Tslpr protein. In some embodiments, the humanized Tslpr protein comprises a signal peptide identical to that of a rodent Tslpr protein (e.g., an endogenous rodent Tslpr protein).

[0027] In some embodiments, the human TSLPR nucleic acid sequence in the humanized Tslpr gene encodes at least a substantial portion of the extracellular domain of the human TSLPR protein. In some embodiments, the human TSLPR nucleic acid sequence in the humanized Tslpr gene encodes amino acids 29-231 of the human TSLPR (e.g., the human TSLPR protein set forth in SEQ ID NO:23). In some embodiments, the human TSLPR nucleic acid sequence includes exon 2 of the human TSLPR gene up to the codon encoding the last extracellular domain amino acid in exon 6.

[0028] In some embodiments, the rodent Tslpr nucleic acid sequence in the humanized Tslpr gene comprises an exon sequence of the rodent Tslpr gene that encodes at least a substantial portion of the transmembrane cytoplasmic sequence of the rodent Tslpr protein (e.g., endogenous rodent Tslpr protein). In some embodiments, the rodent is a mouse, and the rodent Tslpr nucleic acid sequence comprises exon 6 through exon 8 from the codon that encodes the first amino acid of the transmembrane domain of the mouse Tslpr gene (e.g., endogenous mouse Tslpr gene).

[0029] In some embodiments, the rodent Tslpr nucleic acid sequence in the humanized Tslpr gene comprises the exon sequence of the rodent Tslpr gene that encodes the signal peptide of the rodent Tslpr protein (e.g., endogenous rodent Tslpr protein).In some embodiments, the rodent is a mouse, and the rodent nucleic acid sequence comprises exon 1 of the mouse Tslpr gene (e.g., endogenous mouse Tslpr gene).

[0030] In some embodiments, the rodent is a mouse, and the humanized Tslpr gene includes (i) exon 1 of the mouse Tslpr gene (e.g., the endogenous mouse Tslpr gene), (ii) exon 2 of the human TSLPR gene up to the codon encoding the last amino acid of the extracellular domain in exon 6, and (iii) exon 6 from the codon encoding the first amino acid of the transmembrane domain of the mouse Tslpr gene up to exon 8.

[0031] In some embodiments, the humanized Tslpr gene is operably linked to a rodent Tslpr promoter, such as an endogenous rodent Tslpr promoter.

[0032] In some embodiments, the humanized Tslpr gene is located at a locus other than the endogenous rodent Tslpr locus. In some embodiments, the humanized Tslpr gene is located at the endogenous rodent Tslpr locus.

[0033] In some embodiments, the humanized Tslpr gene is located at endogenous rodent Tslpr locus, the humanized Tslpr gene is formed as a result of the replacement of rodent Tslpr genomic DNA with human TSLPR nucleic acid at endogenous rodent Tslpr locus.In some embodiments, the humanized Tslpr gene is formed as a result of the replacement of rodent genomic DNA comprising exon sequence encoding at least a substantial part of the extracellular domain of endogenous rodent Tslpr protein with human TSLPR nucleic acid encoding at least a substantial part of the extracellular domain of human TSLPR protein.In some embodiments, the rodent is a mouse, and the mouse genomic DNA that is replaced comprises exon 2 of endogenous mouse Tslpr gene up to the codon that encodes the last amino acid of the extracellular domain in exon 6, and the human genomic DNA comprises exon 2 of human TSLPR gene up to the codon that encodes the last amino acid of the extracellular domain in exon 6.

[0034] In some embodiments, the rodent is heterozygous for the humanized Tslpr gene. In some embodiments, the rodent is heterozygous for the humanized Tslpr gene.

[0035] In some embodiments, the humanized Tslpr polypeptide is expressed from a humanized Tslpr gene in a rodent.

[0036] In some embodiments, the rodent further comprises in its genome a humanized Tslp gene, a humanized Il7ra gene, or a combination thereof.

[0037] In some embodiments, the rodent is a mouse or a rat.

[0038] In some embodiments, disclosed herein is an isolated rodent cell or tissue, the genome of which comprises a humanized Tslpr gene as described herein.In some embodiments, the rodent cell is a rodent embryonic stem cell.In some embodiments, the rodent cell is an egg or sperm.In some embodiments, the isolated rodent tissue or cell is a mouse tissue or cell, or a rat tissue or cell.

[0039] Disclosed herein, in some embodiments, is a rodent embryo comprising a rodent embryonic stem cell comprising a humanized Tslpr gene as described herein.

[0040] In some embodiments, the method of making genetically modified rodent is disclosed herein.In some embodiments, the method includes: modifying rodent genome to contain humanized Tslpr gene, wherein the humanized Tslpr gene comprises rodent Tslpr nucleic acid sequence and human TSLPR nucleic acid sequence, and encodes humanized Tslpr polypeptide comprising extracellular domain substantially identical to the extracellular domain of human TSLPR protein; and making the rodent that comprises the modified rodent genome.

[0041] In some embodiments, modifying the rodent genome comprises introducing a nucleic acid molecule comprising a human TSLPR nucleic acid sequence into the genome of a rodent embryonic stem (ES) cell; obtaining a rodent ES cell in which the human TSLPR nucleic acid sequence is integrated into the endogenous rodent Tslpr locus to replace the rodent Tslpr genomic DNA, thereby forming a humanized Tslpr gene; and generating a rodent from the obtained rodent ES cell. In some embodiments, the human TSLPR nucleic acid sequence encodes at least a substantial portion of the extracellular domain of the human TSLPR protein. In some embodiments, the nucleic acid molecule introduced into the ES cell further comprises 5' and 3' homology arms flanking the human TSLPR nucleic acid sequence, the 5' and 3' homology arms being homologous to the nucleic acid sequence of the endogenous rodent locus flanking the rodent Tslpr genomic DNA to be replaced. In some embodiments, the humanized Tslpr gene is operably linked to a rodent Tslpr promoter, for example, an endogenous rodent Tslpr promoter at the endogenous rodent Tslp locus.

[0042] In some method embodiments, the rodent is a mouse or a rat.

[0043] In some embodiments, a targeting nucleic acid construct is disclosed herein, comprising a human TSLPR nucleic acid sequence flanked by 5' and 3' nucleotide sequences that are homologous to the nucleotide sequences at the rodent Tslpr locus, which is integrated into a rodent Tslpr gene at an endogenous rodent Tslpr locus, and the integration of the human TSLPR nucleic acid sequence into the rodent Tslpr gene results in the replacement of the rodent Tslpr genomic DNA with the human TSLPR nucleic acid sequence, thereby forming a humanized Tslpr gene, and the human TSLPR nucleic acid sequence encodes at least a substantial portion of the extracellular domain of the human TSLPR protein. In some targeting nucleic acid embodiments, the rodent is a rat or a mouse.

[0044] In some embodiments, an in vitro method for generating genetically modified rodent cells is disclosed herein, comprising introducing into rodent cells a targeting vector comprising a human TSLPR nucleic acid sequence encoding at least a substantial portion of the extracellular domain of the human TSLPR protein, flanked by rodent homology arms that mediate the integration of the human TSLPR nucleotide sequence into endogenous rodent Tslpr locus, resulting in replacement of rodent Tslpr genomic DNA with the human TSLPR nucleic acid sequence, forming the humanized Tslpr gene described herein, thereby generating genetically modified rodent cells. In some embodiments, the rodent cells are mouse or rat cells. In some embodiments, the rodent cells are rodent ES cells, and the method generates genetically modified rodent ES cells.

[0045] In some embodiments, disclosed herein is a genetically modified rodent comprising a humanized Il7ra gene in its genome, wherein the humanized Il7ra gene comprises a rodent Il7ra nucleic acid sequence and a human IL7RA nucleic acid sequence, and wherein the humanized Il7ra gene encodes a humanized Il7ra polypeptide comprising an extracellular domain substantially identical to the extracellular domain of a human IL7RA protein.

[0046] In some embodiments, the humanized Il7ra protein comprises a transmembrane cytoplasmic sequence substantially identical to the transmembrane cytoplasmic sequence of a rodent Il7ra protein (e.g., an endogenous rodent Il7ra protein). In some embodiments, the humanized Il7ra protein comprises a transmembrane cytoplasmic sequence substantially identical to the transmembrane cytoplasmic sequence of a rodent Il7ra protein (e.g., an endogenous rodent Il7ra protein).

[0047] In some embodiments, the humanized Il7ra protein comprises a signal peptide substantially identical to that of a rodent Il7ra protein. In some embodiments, the humanized Il7ra gene comprises a signal peptide identical to that of a rodent Il7ra protein (e.g., an endogenous rodent Il7ra protein).

[0048] In some embodiments, the human IL7RA nucleic acid sequence in the humanized Il7ra gene encodes at least a substantial portion of the extracellular domain of a human IL7RA protein. In some embodiments, the human IL7RA nucleic acid sequence in the humanized Il7ra gene encodes amino acids 21-236 of a human IL7RA protein (e.g., a human IL7RA protein set forth in SEQ ID NO:43). In some embodiments, the human IL7RA nucleic acid sequence includes the codon in exon 1 that encodes the first amino acid of the mature protein of the human IL7RA gene through exon 5.

[0049] In some embodiments, the rodent Il7ra nucleic acid sequence in the humanized Il7ra gene comprises a sequence of a rodent Il7ra gene that encodes at least a substantial portion of the transmembrane cytoplasmic sequence of a rodent Il7ra protein (e.g., an endogenous rodent Il7ra protein). In some embodiments, the rodent is a mouse, and the rodent Il7ra nucleic acid sequence comprises exon 6 through exon 8 of the mouse Il7ra gene (e.g., an endogenous mouse Il7ra gene).

[0050] In some embodiments, the rodent Il7ra nucleic acid sequence in the humanized Il7ra gene comprises a portion of exon 1 of the rodent Il7ra gene that encodes a signal peptide of a rodent Il7ra protein (e.g., an endogenous rodent Il7ra protein). In some embodiments, the rodent is a mouse, and the rodent nucleic acid sequence comprises both the 5'UTR and a portion of exon 1 of the mouse Il7ra gene (e.g., an endogenous mouse Il7ra gene) that encodes the signal peptide of mouse Il7ra.

[0051] In some embodiments, the rodent is a mouse, and the humanized Il7ra gene includes (i) a portion of exon 1 of the mouse Il7ra gene (e.g., the endogenous mouse Il7ra gene), which encodes the signal peptide of the mouse Il7ra protein, (ii) exon 1 from the codon encoding the first amino acid of the mature protein to exon 5 of the human IL7RA gene, and (iii) exon 6 through exon 8 of the mouse Il7ra gene.

[0052] In some embodiments, the humanized Il7ra gene is operably linked to a rodent Il7ra promoter, such as an endogenous rodent Il7ra promoter.

[0053] In some embodiments, the humanized l7ra gene is located at a locus other than the endogenous rodent l7ra locus. In some embodiments, the humanized Il7ra gene is located at the endogenous rodent Il7ra locus.

[0054] In some embodiments, the humanized Il7ra gene is located at the endogenous rodent Il7ra locus, and the humanized Il7ra gene is formed as a result of replacing rodent Il7ra genomic DNA at the endogenous rodent Il7ra locus with a human IL7RA nucleic acid. In some embodiments, the humanized Il7ra gene is formed as a result of replacing rodent genomic DNA comprising an exon sequence encoding at least a substantial portion of the extracellular domain of an endogenous rodent Il7ra protein with a human IL7RA nucleic acid encoding at least a substantial portion of the extracellular domain of a human IL7RA protein. In some embodiments, the rodent is a mouse, and the replaced mouse genomic DNA comprises the codon in exon 1 encoding the first amino acid of the mature protein sequence of the endogenous mouse Il7ra gene through exon 5, and the human genomic DNA comprises the codon in exon 1 encoding the first amino acid of the mature protein sequence of the human IL7RA gene through exon 5.

[0055] In some embodiments, the rodent is heterozygous for the humanized Il7ra gene. In some embodiments, the rodent is homozygous for the humanized Il7ra gene.

[0056] In some embodiments, the humanized Il7ra polypeptide is expressed in a rodent from a humanized Il7ra gene.

[0057] In some embodiments, the rodent further comprises a humanized Tslp gene, a humanized Tslpr gene, or a combination thereof in its genome.

[0058] In some embodiments, the rodent is a mouse or a rat.

[0059] In some embodiments, disclosed herein is an isolated rodent cell or tissue comprising in its genome a humanized Il7ra gene as described herein. In some embodiments, the rodent cell is a rodent embryonic stem cell. In some embodiments, the rodent cell is an egg or sperm. In some embodiments, the isolated rodent tissue or cell is a mouse tissue or cell, or a rat tissue or cell.

[0060] Disclosed herein, in some embodiments, is a rodent embryo comprising a rodent embryonic stem cell comprising a humanized Il7ra gene described herein.

[0061] In some embodiments, a method of generating a genetically modified rodent is disclosed herein. In some embodiments, the method includes modifying a rodent genome to include a humanized Il7ra gene, the humanized Il7ra gene including a rodent Il7ra nucleic acid sequence and a human IL7RA nucleic acid sequence, and encoding a humanized Il7ra polypeptide including an extracellular domain substantially identical to an extracellular domain of a human IL7RA protein, and generating a rodent including the modified rodent genome.

[0062] In some embodiments, modifying the rodent genome comprises introducing a nucleic acid molecule comprising a human IL7RA nucleic acid sequence into the genome of a rodent embryonic stem (ES) cell, obtaining a rodent ES cell in which the human IL7RA nucleic acid sequence is integrated into an endogenous rodent Il7ra locus to replace the rodent Il7ra genomic DNA, thereby forming a humanized Il7ra gene, and using the resulting rodent ES cell to generate a rodent. In some embodiments, the human IL7RA nucleic acid sequence encodes at least a substantial portion of the extracellular domain of a human IL7RA protein. In some embodiments, the nucleic acid molecule introduced into the ES cell further comprises 5' and 3' homology arms flanking the human IL7RA nucleic acid sequence, the 5' and 3' homology arms being homologous to the nucleic acid sequence at the endogenous rodent locus flanking the rodent Il7ra genomic DNA being replaced. In some embodiments, the humanized Il7ra gene is operably linked to a rodent Il7ra promoter, e.g., an endogenous rodent Il7ra promoter at an endogenous rodent Il7ra locus.

[0063] In some method embodiments, the rodent is a mouse or a rat.

[0064] In some embodiments, disclosed herein are targeting nucleic acid constructs comprising a human IL7RA nucleic acid sequence integrated into a rodent Il7ra gene at an endogenous rodent Il7ra locus and flanked by 5' and 3' nucleotide sequences that are homologous to nucleotide sequences at the rodent Il7ra locus, where integration of the human IL7RA nucleic acid sequence into the rodent Il7ra gene results in replacement of rodent Il7ra genomic DNA with the human IL7RA nucleic acid sequence, thereby forming a humanized Il7ra gene, where the human IL7RA nucleic acid sequence encodes at least a substantial portion of the extracellular domain of the human IL7RA protein. In some targeting nucleic acid embodiments, the rodent is a rat or a mouse.

[0065] In some embodiments, an in vitro method for generating a genetically modified rodent cell is disclosed herein, comprising introducing into a rodent cell a targeting vector comprising a human IL7RA nucleic acid sequence encoding at least a substantial portion of the extracellular domain of the human IL7RA protein flanked by rodent homology arms that mediate integration of the human IL7RA nucleotide sequence into an endogenous rodent Il7ra locus, resulting in replacement of the rodent Il7ra genomic DNA with the human IL7RA nucleic acid sequence to form a humanized Il7ra gene as described herein, thereby generating a genetically modified rodent cell. In some embodiments, the rodent cell is a mouse cell or a rat cell. In some embodiments, the rodent cell is a rodent ES cell, and the method generates a genetically modified rodent ES cell.

[0066] In some embodiments, the rodents disclosed herein comprise one or more additional genetic modifications within their genome, such as a humanized Sirpa gene, a disruption of the endogenous RAG2 gene, a disruption of the endogenous IL-2RG gene, a humanized Tpo gene, and a humanized GM-CSF / IL-3 locus. The rodents can be heterozygous or homozygous for any of such additional genetic modifications.

[0067] In some embodiments, a rodent disclosed herein comprises in its genome a humanized Tslp gene and a humanized Sirpα gene, and is homozygous null for both the RAG2 gene and the IL-2RG gene. In some such embodiments, the rodent further comprises in its genome a humanized Tpo gene and / or a humanized GM-CSF / IL-3 locus. The rodent can be heterozygous or homozygous for the humanized genes.

[0068] In some embodiments, the rodent disclosed herein comprises in its genome a humanized Tslp gene, a humanized Tslpr gene, and a humanized Sirpα gene, and is homozygous null for both the RAG2 gene and the IL-2RG gene. In some such embodiments, the rodent further comprises in its genome a humanized Tpo gene and / or a humanized GM-CSF / IL-3 locus. The rodent can be homozygous or heterozygous for the humanized genes.

[0069] In some embodiments, the rodent disclosed herein comprises in its genome a humanized Tslp gene, a humanized Tslpr gene, a humanized Il7ra gene, and a humanized Sirpα gene, and is homozygous null for both the RAG2 gene and the IL-2RG gene. In some such embodiments, the rodent further comprises in its genome a humanized Tpo gene and / or a humanized GM-CSF / IL-3 locus. The rodent can be homozygous or heterozygous for the humanized genes.

[0070] In some embodiments, a genetically modified rodent comprising a humanized Tslp gene, a humanized Tslpr gene, a humanized Il7ra gene, or a combination thereof, as disclosed herein, and optionally having one or more additional genetic modifications, is used in the preparation of a rodent model of allergic disease (e.g., airway or skin inflammation) or cancer.

[0071] In some embodiments, disclosed herein is a method of testing a candidate agent for treating an allergic condition, the method comprising inducing an allergic condition in a genetically modified rodent animal disclosed herein, administering a candidate agent to the rodent animal, and determining whether the candidate agent inhibits the allergic condition in the rodent animal.

[0072] In some embodiments, disclosed herein is a method of testing a candidate agent for treating cancer, the method comprising implanting human cancer cells into a genetically modified rodent as disclosed herein, administering a candidate agent to the rodent, and determining whether the candidate agent inhibits the growth of cancer cells in the rodent. In some embodiments, the cancer is a Th2 driven cancer, including, for example, breast cancer, lung cancer, and pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The patent or application file contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

[0074] The accompanying drawings, which are incorporated in and form a part of this specification, illustrate several embodiments and, together with the description, illustrate the compositions and methods of the present disclosure. [Brief description of the drawings]

[0075] [Figure 1-1] 1 illustrates an exemplary embodiment of a strategy for humanization of the mouse Tslp locus. The mouse Tslp gene and the human TSLP gene are represented by horizontal lines and their exons are represented by boxes located above the lines. A 3486 bp continuous mouse Tslp genomic fragment starting from the codon in exon 2 encoding amino acid 20 in the endogenous mouse Tslp locus to the stop codon in exon 5 has been replaced by a 4100 bp human TSLP genomic fragment starting from the codon encoding amino acid 29 in human TSLP exon 1 to the stop codon in exon 4. The replacement results in a mouse-human hybrid ("humanized") Tslp protein comprising the mouse Tslp signal peptide and the human mature TSLP polypeptide. See also Figures 1D and 1F. [Figure 1-2]Exemplary embodiments of a nucleic acid comprising the mouse Tslp locus and a targeting nucleic acid construct comprising a humanized mouse Tslp locus are illustrated (not to scale), which is a strategy for humanization of the mouse TSLP locus, in which a 3.49 kB mouse genomic fragment at the mouse Tslp locus has been replaced with a humanized fragment comprising a 1.9 kb human genomic fragment 1 (from the codon in exon 1 encoding amino acid 29 to 257 bp after the 3' end of exon 3 of human TLSP), a 4.4 kb Floxed HUb-Puro cassette (inserted into human TLSP intron 3), and a 2.2 kb human genomic fragment 2 (from 258 bp after the end of exon 3 to the stop codon in exon 4 of human TLSP). The humanized fragment in the targeting construct is flanked by a 114.3 kb mouse 5' homology arm and a 65.3 kb mouse 3' homology arm. The targeting nucleic acid construct comprising the humanized mouse Tslp locus can be introduced into mouse embryonic stem cells for targeted insertion into the mouse Tslp gene in the mouse genome.The positions of the primers and probes used in human allele gain assay and mouse allele loss assay to confirm correct targeting are also shown.The sequences of the primers and probes are listed in Table 6. [Figure 1-3] An exemplary embodiment of a strategy for humanization of a mouse TSLP allele is shown (not to scale) in Figure 1B, which illustrates the humanized Tslp allele designated MAID#7466 resulting from replacement of the mouse Tslp genomic fragment described above in with a humanized fragment comprising human fragment 1, a Floxed HUb-Puro cassette, and human genomic fragment 2. After the Floxed HUb-Puro cassette is removed, the humanized Tslp allele is designated MAID#7467. [Figure 1-4] Exemplary embodiments of the protein sequences of mouse Tslp (SEQ ID NO:1), human TSLP isoform 1 (SEQ ID NO:3) (bold italics), and humanized (hybrid) Tslp (SEQ ID NO:5) (human portions in bold italics) are shown, with the signal peptide underlined in each protein sequence. [Figure 1-5]Exemplary embodiments of the mRNA sequences of mouse Tslp (SEQ ID NO:2), human TSLP isoform 1 (SEQ ID NO:4) (bold italics), and humanized (hybrid) Tslp (SEQ ID NO:6) (human portions in bold italics) are shown. The signal peptide-encoding portion of each mRNA is underlined. [Figure 1-6] FIG. 1 shows an alignment of exemplary embodiments of mouse Tslp ("mTslp", SEQ ID NO:1), human TSLP isoform 1 ("hTSLP", SEQ ID NO:3), and humanized (hybrid) Tslp (SEQ ID NO:5) protein sequences. The signal peptides of the proteins are boxed. The junctions between the mouse and human sequences in forming the humanized (hybrid) Tslp protein are indicated by arrows at the 5' (N-terminus) and 3' (C-terminus) of the molecules. The triangle represents the position of human intron 3 (2429 bp) where the Floxed HUb-Puro cassette is inserted. [Figure 1-7] In an exemplary embodiment, mice heterozygous for the Tslp humanization described in Example 1 expressed mature human TSLP protein in mouse serum (center), along with mice without Tslp humanization as a negative control (left) and normal human serum as a positive control (right). Each dot represents one mouse. [Figure 2-1]1 illustrates an exemplary embodiment of a strategy for humanization of the mouse Tslpr locus. The mouse Tslpr gene and the human TSLPR gene are represented by horizontal lines, and their exons are represented by boxes located above the lines. A 2362 bp continuous mouse Tslpr genomic fragment in the endogenous mouse Tslpr locus, which starts in intron 1 328 bp before exon 2 and ends at the 47th bp of exon 6, is replaced by a 13743 bp human TSLPR genomic fragment, which starts in intron 1 909 bp before exon 2 and ends at the 47th bp of exon 6. This substitution results in the deletion of amino acids 27-243 of mouse Tslpr, while preserving the mouse Tslpr signal peptide (amino acids 1-19), the first 7 amino acids of the mouse Tslpr mature protein, and the mouse Tslpr transmembrane domain (amino acids 244-264) and intracellular domain, and inserts a substantial portion of the human TSLPR extracellular domain (starting at amino acid 27 and ending at amino acid 231, just before the human transmembrane domain at amino acids 232-252). See also FIG. 2D. [Figure 2-2]Exemplary embodiments of nucleic acids comprising the mouse Tslpr locus and targeting nucleic acid constructs comprising the humanized mouse Tslpr locus are illustrated (not to scale), which are strategies for humanization of the mouse Tslpr locus, in which a 2.36 kB mouse genomic fragment at the mouse Tslpr locus is replaced by a humanized fragment comprising a 4.8 kb Floxed HUb-Neo cassette (inserted into human TLSP intron 3) and a 13.7 kb human genomic fragment (the 3' portion of intron 1, exon 2 to bp 47 of exon 6 of human TLSP). The humanized fragment in the targeting construct is flanked by a 29.1 kb mouse 5' homology arm (up to 328 bp before exon 2 of mouse Tslpr) and a 133.2 kb mouse 3' homology arm (bp 48 to exon 8 in exon 6 of mouse Tslpr, followed by mouse 3' genomic sequence). The targeting nucleic acid construct comprising humanized mouse Tslpr locus can be introduced into mouse embryonic stem cells for targeted insertion into mouse genome.The positions of primers and probes used in human allele gain assay and mouse allele loss assay to confirm correct targeting are also shown.The sequences of primers and probes are listed in Table 9. [Figure 2-3] An exemplary embodiment of a strategy for humanizing a mouse TSLP allele is shown (not to scale) in FIG. 2B, which illustrates the replacement of the mouse Tslpr genomic fragment described above with a humanized fragment comprising a 4.8 kb Floxed HUb-Neo cassette and a 13743 bp human TSLPR genomic fragment, resulting in a humanized Tslpr allele designated MAID#7558. After the Floxed HUb-Neo cassette is removed, the humanized Tslpr allele is designated MAID#7559. [Figure 2-4] Exemplary embodiments of the protein sequences of mouse Tslpr (SEQ ID NO:21), human TSLPR (SEQ ID NO:23) (bold italics), and humanized (hybrid) Tslpr (SEQ ID NO:25) (human portions in bold italics) are shown. In each protein sequence, the signal peptide ("SP") and transmembrane segment ("TM") are underlined. [Figure 2-5]Exemplary embodiments of the mRNA sequences of mouse Tslpr (SEQ ID NO:22), human TSLPR (SEQ ID NO:24) (bold italics), and humanized (hybrid) Tslpr (SEQ ID NO:26) (human portions in bold italics) are shown. In each mRNA, the portions encoding the signal peptide and transmembrane segment are underlined, respectively. [Figure 2-6] Same as above. [Figure 2-7] FIG. 2 shows an alignment of exemplary embodiments of mouse Tslpr ("mTslpr", SEQ ID NO:21), human TSLPR ("hTSLPR", SEQ ID NO:23), and humanized (hybrid) Tslpr (SEQ ID NO:25) protein sequences. The signal peptide of the proteins is surrounded by a dashed line. The transmembrane domain is surrounded by a solid line. The junctions between the mouse and human sequences in forming the humanized (hybrid) Tslpr are indicated by triangles at the 5' (within intron 1) and 3' (within exon 6) of the molecule. [Figure 3-1] Illustrates an exemplary embodiment of a strategy for humanization of the mouse Il7ra locus. The mouse Il7ra and human IL7RA genes are represented by horizontal lines and their exons are represented by boxes located above the lines. A 19235 bp continuous mouse Il7ra genomic fragment, starting from bp 69 in the coding sequence of exon 1 to the 5' portion of intron 5, in the endogenous mouse Il7ra locus is replaced by a 17232 bp human IL7RA genomic fragment, including bp 69 in the coding sequence of exon 1 to the 5' portion of intron 5. The replacement generates a mouse-human hybrid ("humanized") Tslp protein, including the mouse IL7ra signal peptide, a substantially human extracellular domain (except for the last two amino acids, Gly-Trp), and the transmembrane and intracellular domains of mouse Il7ra. See also FIG. 3D. [Figure 3-2]1 illustrates an exemplary embodiment of a nucleic acid comprising the mouse IL7ra locus and a targeting nucleic acid construct comprising a humanized mouse IL7ra locus (not to scale), a strategy for humanization of the mouse IL7ra locus, in which a 19.2 kB mouse genomic fragment at the mouse IL7ra locus has been replaced with a humanized fragment comprising a 126 bp human genomic fragment 1 (comprising the last 14 bp of exon 1 and the first 112 bp of intron 1 of human IL7RA), a 5.2 kb Floxed HUb-Hyg cassette (inserted into human IL7RA intron 1), and a 17106 bp human genomic fragment 2 (comprising the 3' portion of intron 1, exon 2 to exon 5, and the 5' portion of intron 5 of human IL7RA). The humanized fragment in the targeting construct is flanked by a 48.8 kb mouse 5' homology arm and a 124.3 kb mouse 3' homology arm. The targeting nucleic acid construct containing the humanized mouse Il7ra locus can be introduced into mouse embryonic stem cells for targeted insertion into the mouse genome.The positions of the primers and probes used in human allele gain assay and mouse allele loss assay to confirm correct targeting are also shown.The sequences of the primers and probes are listed in Table 12. [Figure 3-3] An exemplary embodiment of a strategy for humanization of a mouse IL7ra allele is shown (not to scale) in FIG. 3B, which illustrates the replacement of the mouse Il7ra genomic fragment described above with a humanized fragment comprising human genomic fragment 1 (126 bp), a Floxed HUb-Hyg cassette, and human genomic fragment 2 (17106 bp), resulting in a humanized Il7ra allele designated MAID#7266. After the Floxed HUb-hyg cassette is removed, the humanized Il7ra allele is designated MAID#7267. [Diagram 3-4] The protein sequences of mouse Il7ra (SEQ ID NO: 41), human IL7RA (SEQ ID NO: 43) (bold italics), and humanized (hybrid) Il7ra (SEQ ID NO: 45) (human portions in bold italics) are shown. In each protein sequence, the signal peptide and transmembrane domains are underlined. [Figure 3-5]Illustrated are exemplary embodiments of the coding sequences (CDS) for mouse Il7ra (SEQ ID NO: 42), human IL7RA (SEQ ID NO: 44) (bold italics), and humanized (hybrid) Il7ra (SEQ ID NO: 46) (human portions in bold italics). The portion of the human IL7RA sequence used for humanization is underlined. The portion of the hybrid Il7ra sequence having human origin is also underlined. [Diagram 3-6] Same as above. [Diagram 3-7] 3A-3E show an alignment of an exemplary embodiment of mouse Il7ra (SEQ ID NO: 41, top) and human IL7RA (SEQ ID NO: 43, bottom) protein sequences. The signal peptide and transmembrane segments of the proteins (boxed) are shown. The junctions between the mouse and human sequences in forming the humanized (hybrid) Il7ra described in FIGS. 3A-3E are indicated by a vertical line at the N-terminus ("5' junction") and a line near the C-terminus of the extracellular domain ("3' junction"). The amino acids of the extracellular domain involved in humanization are highlighted (starting with the amino acid immediately following the 5' junction and ending at the 3' junction). Triangles represent the junctions of exons within the coding sequence. [Figure 4-1]Mice were sensitized intraperitoneally with either saline and alum (alum only) or ovalbumin and alum (Ova-alum) on days 0 and 14, followed by four consecutive intranasal challenges with Ova on days 21-24. Lung tissue and serum were collected on day 25 for further analysis. 4A. Assessment of pulmonary cellular infiltration by flow cytometry. Lung tissue eosinophil cell frequency is plotted as frequency of total live cells. 4B. Assessment of Muc5a cM RNA expression levels measured by real-time qPCR and expressed relative to β2m (β2-microglobulin) control mRNA expression. ELISA analysis of serum Ova-specific IgE (4C) and Ova-specific IgG1 (4D). Each point represents one mouse. Symbols represent statistical significance compared to saline control (*). 4E illustrates the experimental scheme: mice were sensitized intraperitoneally with either saline and alum (alum only) or ovalbumin and alum (Ova-alum) on days 0 and 14, followed by four consecutive intranasal challenges with Ova on days 21 to 24. On day 25, lung tissue and serum were collected for further analysis of the parameters shown in 4A-4D. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Figure 4-5] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] Disclosed herein are rodents (including, but not limited to, mice and rats) genetically modified to include a humanized Tslp gene, a humanized Tslpr gene, a humanized Il7ra gene, or a combination thereof. The rodents disclosed herein can be used, for example, but not limited to, as models of Th2-driven allergic disease or as models of inflammatory Th2-driven cancer. Compositions and methods for making such genetically modified rodents, as well as methods for using such genetically modified rodents to test candidate therapeutic agents for the treatment of allergies or cancer, are provided and further described below.

[0077] Tslp humanized rodents Thymic stromal lymphopoietin (TSLP) is a member of the four-helix bundle cytokine family and is the distal paralog of interleukin-7 (IL-7). TSLP was first discovered in the culture supernatant of a mouse thymic stromal cell line and has been demonstrated to act as a growth factor for T and B cells. See, e.g., Tsilingiri et al., Cell Mol. Gastroenterology & Hepatology 2017;3:174-182, which is incorporated herein by reference in its entirety. TSLP-expressing cells include epithelial cells, keratinocytes, fibroblasts, stromal cells, dendritic cells, mast cells, and basophils. See Tsilingiri et al. (2017), supra. Two TSLP isoforms exist in humans: a long TSLP isoform (isoform 1) that is expressed at low / undetectable levels at steady state, upregulated during inflammation in multiple tissues, and characterized by exacerbated Th2 responses in multiple Th2-associated diseases (e.g., but not limited to, atopic dermatitis, asthma, allergic reactions, and certain types of cancer), and a short TSLP isoform (isoform 2) that is constitutively expressed from a separate promoter and mediates specific immune homeostatic functions in the intestine and thymus. See Tsilingiri et al. (2017), supra. Unless otherwise indicated, exon numbering of the human TSLP gene is based on the exons encoding the long isoform (human TSLP protein isoform 1).

[0078] Exemplary sequences, including nucleic acid and protein sequences, of human TSLP isoform 1, mouse Tslp, rat Tslp, and humanized Tslp are disclosed in the Sequence Listing and summarized in Table 1. The mouse and rat Tslp genes have a small coding exon 1 and a total of five exons, instead of four exons as in the human TSLP gene. An alignment of the human TSLP isoform 1, mouse Tslp, and humanized (hybrid) Tslp protein sequences is provided in Figure IF. [Table 1]

[0079] In some embodiments, the rodents disclosed herein comprise a humanized Tslp gene in their germline.

[0080] In some embodiments, a rodent disclosed herein comprises a humanized Tslp gene comprising the nucleotide sequence of a rodent Tslp gene and the nucleotide sequence of a human TSLP gene in its genome. As used herein, "nucleotide sequence of a gene" includes the genomic, mRNA, or cDNA sequence of all or a portion of a gene. For example, the nucleotide sequence of a human TSLP gene may be the genomic, mRNA, or cDNA sequence of all or a portion of a human TSLP gene, and the nucleotide sequence of a rodent Tslp gene may be the genomic, mRNA, or cDNA sequence of all or a portion of a rodent Tslp gene (e.g., an endogenous rodent Tslp gene). The nucleotide sequence of a rodent Tslp gene and the nucleotide sequence of a human TSLP gene are operably linked to each other such that the humanized Tslp gene in the rodent genome encodes a humanized Tslp protein that performs the function of a Tslp protein, e.g., binding to the Tslp receptor (Tslpr).

[0081] As used herein, the "human TSLP" gene and protein refer to the TSLP gene and protein of human origin.

[0082] In some embodiments, the human TSLP protein comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the human TSLP protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the human TSLP protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the human TSLP protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 3.

[0083] As used herein, "rodent Tslp" genes and proteins refer to Tslp genes and proteins of rodent (eg, mouse or rat) origin.

[0084] In some embodiments, the mouse Tslp protein is In some embodiments, the mouse Tslp protein comprises the amino acid sequence of SEQ ID NO: 1. Contains an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1. In embodiments, the mouse Tslp protein is It comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mouse Tslp protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:1.

[0085] In some embodiments, the rat Tslp protein comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the rat Tslp protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the rat Tslp protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the rat Tslp protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 7.

[0086] In some embodiments, the genetically modified rodent comprises a humanized Tslp gene in its genome, which encodes a humanized Tslp protein that comprises a mature protein sequence that is substantially identical to the mature protein sequence of a human TSLP protein (such as the human TSLP protein as set forth in SEQ ID NO:3).

[0087] "Mature protein" refers to the portion of a protein after the N-terminal signal peptide has been cleaved.

[0088] A mature protein sequence that is substantially identical to the mature protein sequence of human TSLP protein can be (i) a polypeptide sequence that is at least 95% identical to the mature protein sequence of human TSLP protein, a polypeptide sequence that is at least 98% identical to the mature protein sequence of human TSLP protein, or a polypeptide sequence that is at least 99% identical to the mature protein sequence of human TSLP protein. A mature protein sequence that is substantially identical to the mature protein sequence of human TSLP protein can be a polypeptide sequence identical to the mature protein sequence of human TSLP protein. A mature protein sequence that is substantially identical to the mature protein sequence of human TSLP protein can alternatively or additionally be (ii) a polypeptide sequence that differs by no more than 5 amino acids from the mature protein sequence of human TSLP protein, a polypeptide sequence that differs by no more than 4 amino acids from the mature protein sequence of human TSLP protein, a polypeptide sequence that differs by no more than 3 amino acids from the mature protein sequence of human TSLP protein, a polypeptide sequence that differs by no more than 2 amino acids from the mature protein sequence of human TSLP protein, or a polypeptide sequence that differs by no more than 1 amino acid from the mature protein sequence of human TSLP protein. A mature protein sequence that is substantially identical to the mature protein sequence of the human TSLP protein can alternatively or additionally be (iii) a polypeptide that differs from the mature protein sequence of the human TSLP protein only at the N- or C-terminal portions of a domain, e.g., by having an addition, deletion, or substitution of amino acids (not to exceed 5 amino acids) at the N- or C-terminal portions of the mature protein, where "the N- or C-terminal portions of the mature protein" means within 5 to 10 amino acids from the N- or C-terminus of the mature protein.A mature protein sequence that is substantially identical to the mature protein sequence of the human TSLP protein may alternatively or additionally be (iv) a polypeptide having one or more of the characteristics described in (i) to (iii) above, e.g., a polypeptide that is at least 95% identical to the mature protein sequence of the human TSLP protein and differs from the mature protein sequence of the human TSLP protein by no more than 5 amino acids only in the N-terminal or C-terminal portions of the domain, or a polypeptide that is at least 98% identical to the mature protein sequence of the human TSLP protein and differs from the mature protein sequence of the human TSLP protein by no more than 3 amino acids only in the N-terminal or C-terminal portions of the domain.

[0089] In some embodiments, the human TSLP protein is human TSLP protein isoform 1 as set forth in SEQ ID NO:3, with amino acids 29-159 constituting the mature protein sequence. Thus, in some embodiments, the genetically modified rodent comprises in its genome a humanized Tslp gene that encodes a humanized Tslp protein comprising a mature protein sequence substantially identical to the amino acid sequence set forth in amino acids 29-159 of SEQ ID NO:3. In some embodiments, the humanized Tslp protein comprises a mature protein sequence comprising amino acids 29-159 of SEQ ID NO:3. In some embodiments, the humanized Tslp protein comprises a mature protein sequence comprising amino acids 30-159 of SEQ ID NO:3. In some embodiments, the humanized Tslp protein comprises a mature protein sequence comprising amino acids 31-159 of SEQ ID NO:3. In some embodiments, the humanized Tslp protein comprises a mature protein sequence comprising amino acids 32-159 of SEQ ID NO:3. In some embodiments, the humanized Tslp protein comprises a mature protein sequence comprising amino acids 29-158 of SEQ ID NO:3. In some embodiments, the humanized Tslp protein comprises a mature protein sequence comprising amino acids 29 to 157 of SEQ ID NO: 3. In some embodiments, the humanized Tslp protein comprises a mature protein sequence comprising amino acids 29 to 156 of SEQ ID NO: 3. In some embodiments, the humanized Tslp protein comprises a mature protein sequence shown in amino acids 29 to 159 of SEQ ID NO: 3.

[0090] In some embodiments, the humanized Tslp gene encodes a humanized Tslp protein that comprises a signal peptide that is substantially identical to a signal peptide of the human TSLP protein. In some embodiments, a signal peptide that is substantially identical to a signal peptide of the human TSLP protein is a signal peptide that is at least 95% identical in sequence to the signal peptide of the human TSLP protein. A signal peptide that is substantially identical to a signal peptide of the human TSLP protein can be a signal peptide that is identical to the signal peptide of the human TSLP protein. Additionally or alternatively, a signal peptide that is substantially identical to a signal peptide of the human TSLP protein can be a signal peptide that differs from the signal peptide of the human TSLP protein by no more than 3 amino acids, no more than 2 amino acids, or no more than 1 amino acid. In certain embodiments, the signal peptide of the human TSLP protein comprises the amino acid sequence set forth at amino acids 1-28 of SEQ ID NO:3.

[0091] In some embodiments, the humanized Tslp gene encodes a humanized Tslp protein that comprises a signal peptide that is substantially identical to a signal peptide of a rodent Tslp protein, such as an endogenous rodent Tslp protein. In some embodiments, a signal peptide that is substantially identical to a signal peptide of a rodent Tslp protein is a signal peptide that is at least 95% identical in sequence to the signal peptide of a rodent Tslp protein, and in some embodiments, a signal peptide that is substantially identical to a signal peptide of a rodent Tslp protein is a signal peptide that is identical in sequence to the signal peptide of a rodent Tslp protein. In some embodiments, a signal peptide that is substantially identical to a signal peptide of a rodent Tslp protein is a signal peptide that differs from the signal peptide of a rodent Tslp protein by no more than three amino acids, in some embodiments, a signal peptide that is substantially identical to a signal peptide of a rodent Tslp protein is a signal peptide that differs from the signal peptide of a rodent Tslp protein by no more than two amino acids, in some embodiments, a signal peptide that is substantially identical to a signal peptide of a rodent Tslp protein is a signal peptide that differs from the signal peptide of a rodent Tslp protein by no more than one amino acid. In certain embodiments, a humanized Tslp protein comprises a signal peptide that is substantially identical to a signal peptide of a mouse Tslp protein, e.g., a signal peptide set forth at amino acids 1-19 of SEQ ID NO:1. In certain embodiments, a humanized Tslp protein comprises a signal peptide that is substantially identical to a signal peptide of a rat Tslp protein, e.g., a rat Tslp protein set forth in SEQ ID NO:7.

[0092] As described above, the humanized Tslp gene in the genome of a genetically modified rodent comprises the nucleotide sequence of the human TSLP gene (the "human TSLP nucleotide sequence") and the nucleotide sequence of the rodent Tslp gene (the "endogenous rodent Tslp nucleotide sequence" endogenous rodent Tslp nucleotide sequence).

[0093] In some embodiments, the human TSLP nucleotide sequence in the humanized Tslp gene encodes at least a substantial portion of the mature protein sequence of the human TSLP protein (e.g., human TSLP protein isoform 1). A "substantial portion" of the mature Tslp protein sequence refers to a polypeptide that is nearly the full length of the mature protein sequence. In some embodiments, a substantial portion of the mature protein sequence refers to a polypeptide that is at least 95% of the full length mature protein sequence, in some embodiments, a substantial portion of the mature protein sequence refers to a polypeptide that is at least 98% of the full length mature protein sequence, and in some embodiments, a substantial portion of the mature protein sequence refers to a polypeptide that is at least 99% of the full length mature protein sequence. In some embodiments, a substantial portion of the mature protein sequence refers to a polypeptide that differs from the mature protein sequence by the absence of not more than 5 amino acids at the N-terminus or C-terminus of the mature protein sequence, in some embodiments, a substantial portion of the mature protein sequence refers to a polypeptide that differs from the mature protein sequence by the absence of not more than 4 amino acids at the N-terminus or C-terminus of the mature protein sequence, in some embodiments, a substantial portion of the mature protein sequence refers to a polypeptide that differs from the mature protein sequence by the absence of not more than 3 amino acids at the N-terminus or C-terminus of the mature protein sequence, in some embodiments, a substantial portion of the mature protein sequence refers to a polypeptide that differs from the mature protein sequence by the absence of not more than 2 amino acids at the N-terminus or C-terminus of the mature protein sequence, in some embodiments, a substantial portion of the mature protein sequence refers to a polypeptide that differs from the mature protein sequence by the absence of not more than 1 amino acid at the N-terminus or C-terminus of the mature protein sequence. In some embodiments, the human TSLP nucleotide sequence in the humanized Tslp gene encodes the mature protein sequence of a human TSLP protein (e.g., human TSLP protein isoform 1, such as human TSLP protein isoform 1 set forth in SEQ ID NO:3). In some embodiments, the human TSLP nucleic acid sequence in the humanized Tslp gene encodes amino acids 29-159 of SEQ ID NO: 3. In some embodiments, the human TSLP nucleic acid sequence in the humanized Tslp gene encodes amino acids 30-159 of SEQ ID NO: 3.In some embodiments, the human TSLP nucleic acid sequence in the humanized Tslp gene encodes amino acids 31-159 of SEQ ID NO:3. In some embodiments, the human TSLP nucleic acid sequence in the humanized Tslp gene encodes amino acids 32-159 of SEQ ID NO:3. In some embodiments, the human TSLP nucleic acid sequence in the humanized Tslp gene encodes amino acids 29-158 of SEQ ID NO:3. In some embodiments, the human TSLP nucleic acid sequence in the humanized Tslp gene encodes amino acids 29-157 of SEQ ID NO:3. In some embodiments, the human TSLP nucleic acid sequence in the humanized Tslp gene encodes amino acids 29-156 of SEQ ID NO:3.

[0094] In some embodiments, the human TSLP nucleic acid sequence in the humanized Tslp gene is a cDNA sequence. In some embodiments, the human TSLP nucleotide sequence is a genomic fragment of the human TSLP gene. In some embodiments, the human TSLP nucleotide sequence is a genomic fragment that includes exon sequences that encode at least a substantial portion of the mature protein sequence of the human TSLP protein. In some embodiments, the human TSLP nucleotide sequence is a genomic fragment that includes exon sequences that encode the mature protein sequence of the human TSLP protein (e.g., human TSLP protein isoform 1, such as human TSLP protein isoform 1 set forth in SEQ ID NO:3). In some embodiments, the human TSLP nucleotide sequence is a genomic fragment of the human TSLP gene, the fragment including exon 1, exon 2, portions of exon 3 that encode the mature protein amino acids, and the coding portion of exon 4 (i.e., up to the stop codon in exon 4).

[0095] In some embodiments, the human TSLP nucleotide sequence is a genomic fragment of the human TSLP gene, which fragment also includes the 3'UTR (3' portion of human TSLP exon 4) of the human TSLP gene.

[0096] In some embodiments, the rodent Tslp nucleotide sequence in the humanized Tslp gene encodes a polypeptide substantially identical to the signal peptide of a rodent Tslp protein (e.g., an endogenous rodent Tslp protein). In some embodiments, a polypeptide substantially identical to the signal peptide of a rodent Tslp protein comprises a polypeptide that is at least 95% identical in sequence to the signal peptide of a rodent Tslp protein. In some embodiments, a polypeptide substantially identical to the signal peptide of a rodent Tslp protein comprises a polypeptide that differs from the signal peptide of a rodent Tslp protein by no more than three amino acids. In some embodiments, a polypeptide substantially identical to the signal peptide of a rodent Tslp protein comprises a polypeptide that differs from the signal peptide of a rodent Tslp protein by no more than two amino acids. In some embodiments, a polypeptide substantially identical to the signal peptide of a rodent Tslp protein comprises a polypeptide that differs from the signal peptide of a rodent Tslp protein by no more than one amino acid. In some embodiments, the rodent Tslp nucleotide sequence in the humanized Tslp gene encodes a signal peptide of a rodent Tslp protein (e.g., an endogenous rodent Tslp protein, e.g., a mouse or rat Tslp protein). In some embodiments, the rodent Tslp nucleotide sequence comprises an exon sequence of a rodent Tslp gene that encodes a signal peptide of a rodent Tslp protein. In some embodiments, the rodent Tslp nucleotide sequence is a mouse Tslp nucleotide sequence, and in some embodiments, the mouse Tslp nucleotide sequence comprises exon 1 and a signal peptide amino acid encoding portion of exon 2 of a mouse Tslp gene (e.g., an endogenous mouse Tslp gene). In some embodiments, the rodent Tslp nucleotide sequence in the humanized Tslp gene comprises the 5'UTR of a rodent Tslp gene (e.g., a mouse or rat Tslp gene, e.g., an endogenous mouse or rat Tslp gene).

[0097] In some embodiments, the humanized Tslp gene is operably linked to a rodent Tslp regulatory sequence, e.g., a 5' transcriptional regulatory sequence such as the promoter and / or enhancer of a rodent Tslp gene, e.g., an endogenous rodent 5' transcriptional regulatory sequence at an endogenous rodent Tslp locus, such that expression of the humanized Tslp gene is under the control of the rodent Tslp 5' regulatory sequence.

[0098] In some embodiments, the humanized Tslp gene is at the endogenous rodent Tslp locus. In some embodiments, the humanized Tslp gene is at a locus other than the endogenous rodent Tslp locus, e.g., as a result of random integration. In some embodiments where the humanized Tslp gene is at a locus other than the endogenous rodent Tslp locus, the rodent is unable to express rodent Tslp protein, e.g., as a result of inactivation (e.g., deletion in whole or in part) of the endogenous rodent Tslp gene.

[0099] In some embodiments where the humanized Tslp gene is at the endogenous rodent Tslp locus, the humanized Tslp gene can result from replacement of the endogenous rodent Tslp gene at the endogenous rodent Tslp locus with the nucleotide sequence of the human TSLP gene.

[0100] In some embodiments, the nucleotide sequence of the human TSLP gene that replaces the genomic fragment of the rodent Tslp gene at the endogenous rodent Tslp locus is a cDNA sequence. In some embodiments, the human TSLP nucleotide sequence that replaces the genomic fragment of the rodent Tslp gene at the endogenous rodent Tslp locus is a genomic fragment of the human TSLP gene. In some embodiments, the human TSLP nucleotide sequence encodes at least a substantial portion of the mature protein sequence of the human TSLP protein. In some embodiments, the human TSLP nucleotide sequence is a genomic fragment of the human TSLP gene that includes all or a portion of the exons of the human TSLP gene that encode at least a substantial portion of the mature protein sequence of the human TSLP protein. In some embodiments, the human genomic fragment includes the mature protein amino acid coding portions of exon 1, exon 2, exon 3, and exon 4 of the human TSLP gene. In some embodiments, the human genomic fragment can further include a 3'UTR portion of exon 4 of the human TSLP gene.

[0101] In some embodiments, the human nucleotide sequence integrated into the endogenous rodent Tslp locus is operably linked to a rodent Tslp nucleotide sequence encoding a polypeptide substantially identical to the signal peptide of the rodent Tslp protein. In some embodiments, the human nucleotide sequence integrated into the endogenous rodent Tslp locus is operably linked to an endogenous rodent Tslp genomic sequence that substantially encodes the signal peptide of the endogenous rodent Tslp protein. In some embodiments, the human nucleotide sequence integrated into the endogenous mouse Tslp locus is operably linked to a mouse Tslp nucleotide sequence encoding a polypeptide substantially identical to the signal peptide of the mouse Tslp protein, and in some such embodiments, the mouse Tslp nucleotide sequence comprises exon 1 and the signal peptide amino acid encoding portion of exon 2 of the mouse Tslp gene (e.g., the endogenous mouse Tslp gene).

[0102] In some embodiments, a genomic fragment comprising an exon sequence encoding the mature protein sequence of an endogenous rodent Tslp protein at the endogenous rodent Tslp locus (e.g., a mouse genomic fragment beginning with a codon in exon 2 encoding the first amino acid of the mature mouse Tslp protein to a stop codon) is replaced with a human genomic fragment of a human TSLP gene comprising an exon sequence encoding the mature protein sequence of a human TSLP protein (e.g., a genomic fragment beginning with a codon in exon 1 encoding the first amino acid of the mature human TSLP protein to a stop codon in exon 4). As a result, a humanized Tslp gene is formed at the endogenous rodent Tslp locus. In some embodiments, a humanized Tslp gene is formed at the endogenous mouse Tslp locus and includes mouse Tslp exon 1, the signal peptide amino acid encoding portion of mouse Tslp exon 2, the codon in human TSLP exon 1 encoding the first amino acid of the mature human TSLP protein through a stop codon in human TSLP exon 4, and the mouse 3'UTR of mouse Tslp exon 5. Such a humanized Tslp gene encodes a humanized Tslp protein comprising the mouse Tslp signal peptide and the mature human TSLP polypeptide.

[0103] In some embodiments, the rodents provided herein are heterozygous for a humanized Tslp gene in their genome. In some embodiments, the rodents provided herein are homozygous for a humanized Tslp gene in their genome.

[0104] In some embodiments, the humanized Tslp gene results in expression of the encoded humanized Tslp protein in the rodent, e.g., in the serum of the rodent. In some embodiments, the humanized Tslp protein is expressed in cells and tissues in which the corresponding rodent Tslp protein is expressed in a control rodent (e.g., a rodent that does not have a humanized Tslp gene), e.g., in epithelial cells and keratinocytes of skin, intestine, lung, and eye tissues, as well as dendritic cells, mast cells, and basophils. See, e.g., Tsilingiri et al. (2017), supra.

[0105] In some embodiments, a rodent disclosed herein is unable to express a rodent Tslp protein, e.g., as a result of inactivation (e.g., deletion in whole or in part) or replacement (in whole or in part) of an endogenous rodent Tslp gene.

[0106] TSLPR humanization TSLP acts through a heterodimer composed of a TSLP-specific chain (termed "TSLPR" or "Tslpr") and the IL7 receptor alpha chain. The TSLPR contains a signal peptide, an extracellular domain ("ECD" or "ectodomain"), a transmembrane domain, and an intracellular (cytoplasmic) domain.

[0107] Exemplary sequences, including nucleic acid and protein sequences of human TSLPR isoform 1, mouse Tslpr isoform 1, rat Tslpr isoform 1, and humanized (mouse-human hybrid) Tslpr, are disclosed in the Sequence Listing and summarized in Table 2. An alignment of human TSLPR isoform 1, mouse Tslpr isoform 1, and humanized (mouse-human hybrid) Tslpr protein sequences is provided in FIG. 2F. Unless otherwise indicated, the numbering of exons in the human and mouse genes is based on exons encoding protein isoform 1 in the human and mouse. [Table 2-1] [Table 2-2]

[0108] In some embodiments, the rodents disclosed herein comprise a humanized Tslpr gene in the germline.

[0109] In some embodiments, the rodent disclosed herein comprises a humanized Tslpr gene, comprising the nucleotide sequence of a rodent Tslpr gene (e.g., an endogenous rodent Tslpr gene) and the nucleotide sequence of a human TSLPR gene in its genome. As used herein, the "nucleotide sequence of a gene" includes the genomic sequence, mRNA sequence or cDNA sequence of all or part of a gene. As a non-limiting example, the nucleotide sequence of a human TSLPR gene includes the genomic sequence, mRNA sequence or cDNA sequence of all or part of a human TSLPR gene. The nucleotide sequence of a rodent Tslpr gene and the nucleotide sequence of a human TSLPR gene are operably linked to each other, such that the humanized Tslpr gene in the rodent genome encodes a humanized Tslpr protein having a Tslpr protein structure (including an extracellular domain, a transmembrane domain and a cytoplasmic domain) and performs a Tslpr function (e.g., binds Tslp protein and forms a heterodimer with IL-7 receptor).

[0110] As used herein, "human TSLPR" genes and proteins refer to TSLPR genes and proteins of human origin. In some embodiments, the human TSLPR protein comprises the amino acid sequence of SEQ ID NO:23. In some embodiments, the human TSLPR protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:23. In some embodiments, the human TSLPR protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:23. In some embodiments, the human TSLPR protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:23.

[0111] As used herein, "rodent Tslpr" genes and proteins refer to Tslpr genes and proteins of rodent (e.g., mouse or rat) origin. In some embodiments, the mouse Tslpr protein comprises the amino acid sequence of SEQ ID NO:21. In some embodiments, the mouse Tslpr protein comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:21. In some embodiments, the mouse Tslpr protein comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:21. In some embodiments, the mouse Tslpr protein comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:21. In some embodiments, the rat Tslpr protein comprises the amino acid sequence of SEQ ID NO:27. In some embodiments, the rat Tslpr protein comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:27. In some embodiments, the rat Tslpr protein comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:27. In some embodiments, the rat Tslpr protein comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:27.

[0112] In some embodiments, the genetically modified rodent contains a humanized Tslpr gene in its genome, which encodes a humanized Tslpr protein containing an extracellular domain substantially identical to the extracellular domain of the human TSLPR protein. In some embodiments, the extracellular domain substantially identical to the extracellular domain of the human TSLPR protein exhibits the same functionality (e.g., ligand binding properties) as the extracellular domain of the human TSLPR protein. An extracellular domain or polypeptide that is "substantially identical to the extracellular domain of the human TSLPR protein" can be (i) a polypeptide that is at least 95% identical in sequence to the extracellular domain of the human TSLPR protein, a polypeptide that is at least 98% identical in sequence to the extracellular domain of the human TSLPR protein, or a polypeptide that is at least 99% identical in sequence to the extracellular domain of the human TSLPR protein. An extracellular domain or polypeptide that is "substantially identical to the extracellular domain of the human TSLPR protein" can be a polypeptide that is 100% identical in sequence to the extracellular domain of the human TSLPR protein. Alternatively or additionally, an extracellular domain or polypeptide that is "substantially identical to the extracellular domain of the human TSLPR protein" can be (ii) a polypeptide that differs by no more than 10 amino acids from the extracellular domain of the human TSLPR protein, a polypeptide that differs by no more than 7 amino acids from the extracellular domain of the human TSLPR protein, a polypeptide that differs by no more than 5 amino acids from the extracellular domain of the human TSLPR protein, a polypeptide that differs by no more than 4 amino acids from the extracellular domain of the human TSLPR protein, a polypeptide that differs by no more than 3 amino acids from the extracellular domain of the human TSLPR protein, a polypeptide that differs by no more than 2 amino acids from the extracellular domain of the human TSLPR protein, or a polypeptide that differs by no more than 1 amino acid from the extracellular domain of the human TSLPR protein.Alternatively or additionally, an extracellular domain or polypeptide that is "substantially identical to the extracellular domain of the human TSLPR protein" may be (iii) a polypeptide that differs from the extracellular domain of the human TSLPR protein only at the N-terminal or C-terminal portion of the extracellular domain, for example, by having amino acid additions, deletions, and / or substitutions at the N-terminal and / or C-terminal portions of the extracellular domain (i.e., within 5-10 amino acids of the N- or C-terminus of the extracellular domain). Alternatively or additionally, an extracellular domain or polypeptide that is "substantially identical to the extracellular domain of the human TSLPR protein" may be (iv) a polypeptide having one or more of the characteristics detailed in (i) to (iii) above, such as a polypeptide that is at least 95% identical in sequence to the extracellular domain of the human TSLPR protein and differs from the extracellular domain of the human TSLPR protein by no more than 10 amino acids only in the N-terminal or C-terminal portion of the extracellular domain, a polypeptide that is at least 95% identical in sequence to the extracellular domain of the human TSLPR protein and differs from the extracellular domain of the human TSLPR protein by no more than 5 amino acids only in the N-terminal or C-terminal portion of the extracellular domain, or a polypeptide that is at least 98% identical in sequence to the extracellular domain of the human TSLPR protein and differs from the extracellular domain of the human TSLPR protein by no more than 3 amino acids only in the N-terminal or C-terminal portion of the extracellular domain. In certain embodiments, the human TSLPR protein comprises the amino acid sequence set forth in SEQ ID NO:23, and its extracellular domain is composed of amino acids 23-231 of SEQ ID NO:23. In some embodiments, the humanized Tslpr gene encodes a humanized Tslpr protein whose extracellular domain is substantially identical to the extracellular domain of the human TSLPR protein set forth in SEQ ID NO:23, i.e., substantially identical to amino acids 23-231 of SEQ ID NO:23.For example, the humanized Tslpr gene encodes a humanized Tslpr protein having an extracellular domain comprising amino acids 23-231, 24-231, 25-231, 26-231, 27-231, 28-231, 23-230, 23-229, 23-228, 23-227, or 23-226 of SEQ ID NO:23. In some embodiments, the humanized Tslpr gene encodes a humanized Tslpr protein having an extracellular domain comprising amino acids 23-231 of SEQ ID NO:23. In some embodiments, the humanized Tslpr gene encodes a humanized Tslpr protein having an extracellular domain comprising amino acids 25-231 of SEQ ID NO:23. In some embodiments, the humanized Tslpr gene encodes a humanized Tslpr protein having an extracellular domain comprising amino acids 27-231 of SEQ ID NO:23. In some embodiments, the humanized Tslpr gene encodes a humanized Tslpr protein having an extracellular domain comprising amino acids 23-228 of SEQ ID NO:23. In some embodiments, the humanized Tslpr gene encodes a humanized Tslpr protein having an extracellular domain comprising amino acids 23-226 of SEQ ID NO:23.

[0113] In some embodiments, the humanized Tslpr gene encodes a humanized Tslpr protein having an extracellular domain that includes amino acids from the N-terminus of the extracellular domain of rodent Tslpr (e.g., endogenous rodent Tslpr), followed by the extracellular domain of human TSLPR or a substantial portion thereof. A "substantial portion of the extracellular domain" of a human TSLPR (or rodent Tslpr) protein refers to a polypeptide that is nearly the complete extracellular domain of the protein. In some embodiments, a substantial portion of the extracellular domain of a human TSLPR protein refers to a polypeptide that is at least 95% of the full-length extracellular domain of a human TSLPR protein. In some embodiments, a substantial portion of the extracellular domain of a human TSLPR protein refers to a polypeptide that is at least 98% of the full-length extracellular domain of a human TSLPR protein. Sequence In some embodiments, a substantial portion of the extracellular domain of a human TSLPR protein refers to a polypeptide that differs from the full-length extracellular domain by the absence of no more than 10 amino acids at the N-terminus or C-terminus of the extracellular domain. In some embodiments, a substantial portion of the extracellular domain of a human TSLPR protein refers to a polypeptide that differs from the full-length extracellular domain by the absence of no more than 5 amino acids at the N-terminus or C-terminus of the extracellular domain. In some embodiments, a substantial portion of the extracellular domain of a human TSLPR protein refers to a polypeptide that differs from the full-length extracellular domain by the absence of no more than 4 amino acids at the N-terminus or C-terminus of the extracellular domain. For example, the extracellular domain of a human TSLPR protein set forth in SEQ ID NO:23 is defined by amino acids 23-231, and examples of substantial portions of the extracellular domain can include amino acids 25-231, 26-231, 27-231, 23-228, 23-227, or 23-226 of SEQ ID NO:23. In some embodiments, a substantial portion of the extracellular domain of a human TSLPR protein includes amino acids 25-231 of SEQ ID NO:23. In some embodiments, a substantial portion of the extracellular domain of a human TSLPR protein includes amino acids 27-231 of SEQ ID NO:23. In some embodiments, the substantial portion of the extracellular domain of the human TSLPR protein comprises amino acids 23-228 of SEQ ID NO:23.In some embodiments, a substantial portion of the extracellular domain of a human TSLPR protein comprises amino acids 23-226 of SEQ ID NO:23. In some embodiments, the extracellular domain of a humanized Tslpr protein comprises 6-8 amino acids from the N-terminus of the extracellular domain of a rodent Tslpr (e.g., endogenous rodent Tslpr), followed by a substantial portion of the extracellular domain of a human TSLPR protein. In some embodiments, the extracellular domain of a humanized Tslpr protein comprises 7 amino acids from the N-terminus of the extracellular domain of a rodent Tslpr (e.g., endogenous rodent Tslpr), followed by amino acids 27-231 of SEQ ID NO:23. In some embodiments, the humanized Tslpr gene encodes a humanized Tslpr protein containing an extracellular domain set forth at amino acids 20-231 of SEQ ID NO:25 - which extracellular domain includes the N-terminal 7 amino acids of the mouse Tslpr extracellular domain followed by amino acids 27-231 of SEQ ID NO:23 (human Tslpr), which differs from the extracellular domain of the human TSLPR protein of SEQ ID NO:23 at the N-terminus of the extracellular domain ("AAAVTSR" (SEQ ID NO:67) in humanized TSLPR of SEQ ID NO:25 as opposed to "QGGA" (SEQ ID NO:68) in human TSLPR of SEQ ID NO:23), but the remaining ame 205 amino acids are identical to the human TSLP extracellular domain.

[0114] In some embodiments, the humanized Tslpr gene encodes a humanized Tslpr protein that contains a transmembrane cytoplasmic sequence (i.e., a sequence that includes both a transmembrane domain and a cytoplasmic domain) that is substantially identical to the transmembrane cytoplasmic sequence of a rodent Tslpr protein, such as an endogenous rodent Tslpr protein. In some embodiments, the transmembrane cytoplasmic sequence that is substantially identical to the transmembrane cytoplasmic sequence of an endogenous rodent Tslpr protein exhibits the same functionality (e.g., signal transduction and / or interaction with intracellular molecules) as the transmembrane cytoplasmic sequence of a rodent Tslpr protein, such as an endogenous rodent Tslpr protein. A transmembrane cytoplasmic sequence or polypeptide that is "substantially identical to a transmembrane cytoplasmic sequence of a rodent Tslpr protein" can be (i) a polypeptide that is at least 95% identical in sequence to a transmembrane cytoplasmic sequence of a rodent Tslpr protein, or a polypeptide that is at least 98% identical in sequence to a transmembrane cytoplasmic sequence of a rodent Tslpr protein. A transmembrane cytoplasmic sequence or polypeptide that is "substantially identical to a transmembrane cytoplasmic sequence of a rodent Tslpr protein" can be a polypeptide that is identical to a transmembrane cytoplasmic sequence of a rodent Tslpr protein. Alternatively or additionally, a transmembrane cytoplasmic sequence or polypeptide that is "substantially identical to the transmembrane cytoplasmic sequence of a rodent Tslpr protein" can be (ii) a polypeptide that differs by no more than 5 amino acids from the transmembrane cytoplasmic sequence of a rodent Tslpr protein, a polypeptide that differs by no more than 4 amino acids from the transmembrane cytoplasmic sequence of a rodent Tslpr protein, a polypeptide that differs by no more than 3 amino acids from the transmembrane cytoplasmic sequence of a rodent Tslpr protein, a polypeptide that differs by no more than 2 amino acids from the transmembrane cytoplasmic sequence of a rodent Tslpr protein, or a polypeptide that differs by no more than 1 amino acid from the transmembrane cytoplasmic sequence of a rodent Tslpr protein.Alternatively or additionally, a transmembrane cytoplasmic sequence or polypeptide that is "substantially identical to a transmembrane cytoplasmic sequence of a rodent Tslpr protein" may be (iii) in some embodiments a polypeptide that differs from the transmembrane cytoplasmic sequence of a rodent Tslpr protein only at its N-terminus or C-terminus, e.g., by having amino acid additions, deletions or substitutions at the N-terminal or C-terminal portions of the transmembrane cytoplasmic sequence. Alternatively or additionally, a transmembrane cytoplasmic sequence or polypeptide that is "substantially identical to the transmembrane cytoplasmic sequence of a rodent Tslpr protein" may be (iv) a polypeptide having one or more of the characteristics detailed in (i) to (iii) above, for example, a polypeptide that is at least 95% identical in sequence to the transmembrane cytoplasmic sequence of a rodent Tslpr protein and differs from the transmembrane cytoplasmic sequence of the rodent Tslpr protein by no more than 5 amino acids only at the N-terminus or C-terminus, or a polypeptide that is at least 95% identical in sequence to the transmembrane cytoplasmic sequence of a rodent Tslpr protein and differs from the transmembrane cytoplasmic sequence of the rodent Tslpr protein by no more than 3 amino acids only at the N-terminus or C-terminus. "N- or C-terminal portion of the transmembrane cytoplasmic sequence" means within 5 to 10 amino acids from the N-terminus of the transmembrane domain, or within 5 to 10 amino acids from the C-terminus of the cytoplasmic domain. In some embodiments, the humanized Tslpr protein contains a transmembrane cytoplasmic sequence that is substantially identical to the transmembrane cytoplasmic sequence of the mouse Tslpr protein (such as the endogenous mouse Tslpr protein). In some embodiments, the humanized Tslpr protein contains a transmembrane cytoplasmic sequence that is substantially identical to the transmembrane cytoplasmic sequence of the rat Tslpr protein (such as the endogenous rat Tslpr protein).

[0115] In some embodiments, the humanized Tslpr gene encodes a humanized Tslpr protein that includes a signal peptide that is substantially identical to the signal peptide of a rodent Tslpr protein (e.g., an endogenous rodent Tslpr protein). A signal peptide that is "substantially identical to the signal peptide of a rodent Tslpr protein" can be (i) a polypeptide that is at least 95% identical in sequence to the signal peptide of a rodent Tslpr protein, or a polypeptide that is identical in sequence to the signal peptide of a rodent Tslpr protein. Alternatively or additionally, a signal peptide that is "substantially identical to the signal peptide of a rodent Tslpr protein" can be (ii) a polypeptide that differs from the signal peptide of an endogenous rodent Tslpr protein by no more than three amino acids, a polypeptide that differs from the signal peptide of an endogenous rodent Tslpr protein by no more than two amino acids, or a polypeptide that differs from the signal peptide of an endogenous rodent Tslpr protein by no more than one amino acid. Alternatively or additionally, a signal peptide that is "substantially identical to a signal peptide of a rodent Tslpr protein" may be a polypeptide that differs from the signal peptide of an endogenous rodent Tslpr protein only at the N-terminus or C-terminus, for example, by having an amino acid addition, deletion or substitution at the N-terminus or C-terminus portion of the signal peptide. Alternatively or additionally, a signal peptide that is "substantially identical to a signal peptide of a rodent Tslpr protein" may be (iv) a polypeptide that has one or more of the characteristics detailed in (i)-(iii) above, for example, a polypeptide that is at least 95% identical to a signal peptide of a rodent Tslpr protein and differs from the signal peptide of a rodent Tslpr protein only by not more than 3 amino acids at the N-terminus or C-terminus. "N- or C-terminal portion of a signal peptide" means within 5 amino acids from the N- or C-terminus of the signal peptide. In some embodiments, the humanized Tslpr protein comprises a signal peptide that is substantially identical to the signal peptide of a mouse Tslpr protein (such as an endogenous mouse Tslpr protein).In some embodiments, the humanized Tslpr protein comprises a signal peptide that is substantially identical to the signal peptide of a rat Tslpr protein (such as an endogenous rat Tslpr protein).

[0116] In some embodiments, the humanized Tslpr gene in the genome of the genetically modified rodent comprises a nucleotide sequence of the human TSLPR gene (the "human TSLPR nucleotide sequence"), and a nucleotide sequence of a rodent Tslpr gene (the "rodent Tslpr nucleotide sequence", e.g., an endogenous rodent Tslpr nucleotide sequence), wherein the human TSLPR nucleotide sequence encodes at least a substantial portion of the extracellular domain of the human TSLPR protein. As discussed above, examples of substantial portions of the extracellular domain of the human TSLPR can include amino acids 25-231, 26-231, 27-231, 23-228, 23-227, or 23-226 of SEQ ID NO:23. In some embodiments, a substantial portion of the extracellular domain comprises amino acids 27-231 of SEQ ID NO:23. In some embodiments, a substantial portion of the extracellular domain comprises amino acids 25-231 of SEQ ID NO:23. In some embodiments, a substantial portion of the extracellular domain comprises amino acids 23-228 of SEQ ID NO:23. In some embodiments, a substantial portion of the extracellular domain comprises amino acids 23-226 of SEQ ID NO:23. In some embodiments, the human TSLPR nucleotide sequence is a cDNA sequence. In some embodiments, the human TSLPR nucleotide sequence in the humanized Tslpr gene encodes the extracellular domain of a human TSLPR protein (e.g., human TSLPR protein isoform 1 as defined in SEQ ID NO:23). In some embodiments, the human TSLPR nucleotide sequence is a genomic fragment of the human TSLPR gene. In some embodiments, the human TSLPR nucleotide sequence is a genomic fragment of the human TSLPR gene comprising exon 2 through the codon of exon 6 that encodes the last amino acid of the extracellular domain of the human TSLPR protein. In some embodiments, the human TSLPR genomic fragment encodes amino acids 27-231 of human TSLPR isoform 1, with the four amino acids missing at the N-terminus of the extracellular domain of human TSLPR isoform 1. In some embodiments, the human TSLPR genomic fragment further comprises a 3' portion of intron 1 operably linked to exon 2 through the codon of exon 6, which encodes the last amino acid of the extracellular domain of the human TSLPR protein.

[0117] In some embodiments, the humanized Tslpr gene in the genome of the genetically modified rodent comprises a rodent Tslpr nucleotide sequence and a human TSLPR nucleotide sequence, and the rodent Tslpr nucleotide sequence encodes a polypeptide substantially identical to the transmembrane cytoplasmic sequence of the rodent Tslpr protein (e.g., endogenous rodent Tslpr protein). In some embodiments, the rodent Tslpr nucleotide sequence present in the humanized Tslpr gene encodes the transmembrane cytoplasmic sequence of the endogenous rodent Tslpr protein. In some embodiments, the rodent Tslpr nucleotide sequence present in the humanized Tslpr gene is a mouse Tslpr nucleotide sequence, and in some embodiments, the mouse Tslpr nucleotide sequence comprises a portion of the mouse Tslpr gene (e.g., endogenous mouse Tslpr gene) from exon 6 (starting with the codon encoding the first amino acid of the mouse Tslpr transmembrane domain) to exon 8.

[0118] In some embodiments, the humanized Tslpr gene in the genome of the genetically modified rodent comprises a rodent Tslpr nucleotide sequence upstream (5') of the human TSLPR nucleotide sequence, and the rodent Tslpr nucleotide sequence encodes a polypeptide substantially identical to a signal peptide of a rodent Tslpr protein (e.g., an endogenous rodent Tslpr protein). In some embodiments, the rodent Tslpr nucleotide sequence encoding a polypeptide substantially identical to a signal peptide of a rodent Tslpr protein is a mouse Tslpr nucleotide sequence (e.g., an endogenous mouse Tslpr nucleotide sequence) or a rat Tslpr nucleotide sequence (e.g., an endogenous rat Tslpr nucleotide sequence). In some embodiments, the rodent Tslpr nucleotide sequence encodes a polypeptide comprising a signal peptide sequence and amino acids (e.g., 6-8 amino acids) from the N-terminus of the extracellular domain of a rodent Tslpr protein. In some embodiments, the rodent Tslpr nucleotide sequence encodes a polypeptide comprising the signal peptide sequence and 7 amino acids from the N-terminus of the extracellular domain of the rodent Tslpr protein. In some embodiments, the rodent Tslpr nucleotide sequence is a mouse Tslpr nucleotide sequence that comprises exon 1 of the mouse Tslpr gene (e.g., an endogenous mouse Tslpr gene), and in some such embodiments, the mouse Tslpr nucleotide sequence further comprises a 5' portion of intron 1 of the mouse Tslpr gene.

[0119] In some embodiments, the humanized Tslpr gene is operably linked to a rodent Tslpr 5' regulatory sequence, such as an endogenous rodent Tslpr regulatory sequence, e.g., a 5' transcription regulatory sequence, such as a promoter and / or enhancer, such that expression of the humanized Tslpr gene is under the control of the rodent Tslpr 5' regulatory sequence.

[0120] In some embodiments, the humanized Tslpr gene is at the endogenous rodent Tslpr locus. In some embodiments, the humanized Tslpr gene is at a locus other than the endogenous rodent Tslpr locus, for example, as a result of random integration. In some embodiments, the humanized Tslpr gene is at a locus other than the endogenous rodent Tslpr locus, for example, as a result of inactivation (e.g., deletion in whole or in part) of the endogenous rodent Tslpr gene, the rodent cannot express rodent Tslpr protein.

[0121] In some embodiments, where the humanized Tslpr gene is at the endogenous rodent Tslpr locus, the humanized Tslpr gene may result from replacement of the nucleotide sequence of an endogenous rodent Tslpr gene at the endogenous rodent Tslpr locus with the nucleotide sequence of a human TSLPR gene.

[0122] In some embodiments, the nucleotide sequence of the endogenous rodent Tslpr gene to be replaced at the endogenous rodent Tslpr locus is a genomic fragment of the endogenous rodent Tslpr gene encoding at least a substantial portion of the extracellular domain of the rodent Tslpr protein. In some embodiments, the rodent is a mouse, and the mouse Tslpr genomic fragment to be replaced encodes at least a substantial portion of the extracellular domain of the endogenous mouse Tslpr protein. For example, the extracellular domain of mouse Tslpr of SEQ ID NO:21 is defined by amino acids 20-243, and examples of substantial portions of the extracellular domain may include amino acids 21-243, 22-243, 23-243, 24-243, 25-243, 26-243, 27-243, 20-241, 20-240, 20-239, and 20-238 of SEQ ID NO:21. In some embodiments, a substantial portion of the extracellular domain of the mouse Tslpr protein comprises amino acids 27-243 of SEQ ID NO:21. In some embodiments, a substantial portion of the extracellular domain of the mouse Tslpr protein comprises amino acids 25-243 of SEQ ID NO:21. In some embodiments, a substantial portion of the extracellular domain of the mouse Tslpr protein comprises amino acids 20-240 of SEQ ID NO:21. In some embodiments, a substantial portion of the extracellular domain of the mouse Tslpr protein comprises amino acids 20-238 of SEQ ID NO:21. In some embodiments, the mouse Tslpr genomic fragment to be replaced comprises exon 2 through the codon of exon 6 that encodes the last amino acid of the extracellular domain.

[0123] In some embodiments, the nucleotide sequence of the human TSLPR gene that replaces the genomic fragment of the rodent Tslpr gene at the endogenous rodent Tslpr locus is a cDNA sequence.In some embodiments, the human TSLPR nucleotide sequence that replaces the genomic fragment of the rodent Tslpr gene at the endogenous rodent Tslpr locus is a genomic fragment of the human TSLPR gene.In some embodiments, the genomic fragment of the human TSLPR gene that replaces the genomic fragment of the rodent Tslpr gene at the endogenous rodent Tslpr locus comprises all or part of the exons of the human TSLPR gene that code for at least a substantial portion of the extracellular domain of the human TSLPR protein. As discussed above, examples of a substantial portion of the extracellular domain of human TSLPR may include amino acids 23-231, 24-231, 25-231, 26-231, 27-231, 28-231, 23-230, 23-229, 23-228, 23-227, or 23-226 of SEQ ID NO:23. In some embodiments, a substantial portion of the extracellular domain of human TSLPR includes amino acids 25-231 of SEQ ID NO:23. In some embodiments, a substantial portion of the extracellular domain of human TSLPR includes amino acids 27-231 of SEQ ID NO:23. In some embodiments, a substantial portion of the extracellular domain of human TSLPR includes amino acids 23-228 of SEQ ID NO:23. In some embodiments, a substantial portion of the extracellular domain of human TSLPR includes amino acids 23-226 of SEQ ID NO:23. In some embodiments, the human genomic fragment comprises human TSLPR exon 2 through exon 6 codons encoding the final amino acid of the human TSLPR extracellular domain.

[0124] In some embodiments, the human TSLPR nucleotide sequence inserted into the endogenous rodent Tslpr locus is operably linked to a genomic sequence of a rodent Tslpr gene that encodes a polypeptide substantially identical to the transmembrane cytoplasmic sequence of a rodent Tslpr protein (e.g., an endogenous rodent, mouse, or rat Tslpr protein). In embodiments where the rodent is a mouse, the genomic sequence of the mouse Tslpr gene, in some embodiments, includes exon 6 from the codon encoding the first amino acid of the transmembrane domain of the mouse Tslpr gene (e.g., an endogenous mouse Tslpr gene).

[0125] In some embodiments, the human TSLPR nucleotide sequence inserted into the endogenous rodent Tslpr locus is operably linked to a genomic sequence of a rodent Tslpr gene that encodes a polypeptide substantially identical to the signal peptide of a rodent Tslpr protein (e.g., an endogenous rodent, mouse or rat Tslpr protein). In embodiments where the rodent is a mouse, the genomic sequence of the mouse Tslpr gene, in some embodiments, includes all or a portion of exon 1, and optionally intron 1, of the mouse Tslpr gene (e.g., an endogenous mouse Tslpr gene).

[0126] In some embodiments, the rodent is a mouse, and at the endogenous mouse Tslpr locus, a genomic fragment of the endogenous mouse Tslpr gene including exon 2 of the mouse Tslpr extracellular domain to the codon in exon 6 that codes for the last amino acid is replaced with a genomic fragment of the human TSLPR gene including exon 2 of the human TSLPR extracellular domain to the codon in exon 6 that codes for the last amino acid. In some embodiments, the humanized Tslpr gene is formed at the endogenous rodent Tslpr locus and includes exon 1 of the mouse Tslpr gene, exon 2 of the human TSLPR gene to the codon in exon 6 that codes for the last amino acid, and exon 6 of the mouse Tslpr gene starting from the codon that codes for the first amino acid of the mouse Tslpr transmembrane domain to exon 8.

[0127] In some embodiments, the rodent provided herein is heterozygous for the humanized Tslpr gene in its genome. In some embodiments, the rodent provided herein is homozygous for the humanized Tslpr gene in its genome.

[0128] In some embodiments, the humanized Tslpr gene causes expression of the encoded humanized Tslpr protein in the rodent. In some embodiments, the humanized Tslpr protein is expressed in cells and tissues, and the corresponding rodent Tslpr protein in a control rodent (e.g., a rodent that does not have the humanized Tslpr gene) is typically expressed in non-immune cell types, such as, for example, dendritic cells, CD4+ T cells, and group 2 innate lymphoid cells, as well as epithelial, endothelial, and smooth muscle cells.

[0129] In some embodiments, a rodent disclosed herein is unable to express a rodent Tslpr protein, e.g., as a result of inactivation (e.g., deletion in whole or in part) or replacement (in whole or in part) of the endogenous rodent Tslpr gene.

[0130] IL7RA humanization TSLP acts through a heterodimer composed of a chain specific for TSLP (termed "TSLPR" or "Tslpr") and the IL7 receptor alpha chain ("IL7RA" for humans and "Il7ra" for non-human or humanized molecules). IL7RA contains a signal peptide, an extracellular domain ("ECD" or "ectodomain"), a transmembrane domain, and an intracellular (cytoplasmic) domain.

[0131] Exemplary sequences, including nucleic acid and protein sequences, of human IL7RA, mouse Il7ra, rat Il7ra, and humanized (mouse-human hybrid) Il7ra are disclosed in the Sequence Listing and summarized in Table 3. An alignment of the human IL7RA and mouse Il7ra protein sequences is presented in Figure 3F. [Table 3-1] [Table 3-2]

[0132] In some embodiments, the rodent disclosed herein comprises a humanized Il7ra gene in the germline.

[0133] In some embodiments, a rodent disclosed herein comprises in its genome a humanized Il7ra gene, comprising the nucleotide sequence of a rodent Il7ra gene (e.g., an endogenous rodent Il7ra gene) and the nucleotide sequence of a human IL7RA gene. As used herein, "nucleotide sequence of a gene" includes the genomic, mRNA, or cDNA sequence of all or a portion of a gene. As a non-limiting example, the nucleotide sequence of a human IL7RA gene includes the genomic, mRNA, or cDNA sequence of all or a portion of a human IL7RA gene. The nucleotide sequence of the rodent Il7ra gene and the nucleotide sequence of the human IL7RA gene are operably linked to each other such that the humanized Il7ra gene in the rodent genome encodes a humanized Il7ra protein having an Il7ra protein structure (including an extracellular domain, a transmembrane domain, and a cytoplasmic domain) and performs an Il7ra function (e.g., binding Il7 protein and forming a heterodimer with Tslpr, which binds Tslp).

[0134] As used herein, "human IL7RA" genes and proteins refer to IL7RA genes and proteins of human origin. In some embodiments, the human IL7RA protein comprises the amino acid sequence of SEQ ID NO:43. In some embodiments, the human IL7RA protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the human IL7RA protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the human IL7RA protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:43.

[0135] As used herein, "rodent Il7ra" genes and proteins refer to Il7rar genes and proteins of rodent (e.g., mouse or rat) origin. In some embodiments, the mouse IL7ra protein comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the mouse IL7ra protein comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the mouse IL7rar protein comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the mouse IL7rar protein comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the rat IL7ra protein comprises the amino acid sequence of SEQ ID NO:47. In some embodiments, the rat IL7ra protein comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:47. In some embodiments, the rat IL7ra protein comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:47. In some embodiments, the rat IL7ra protein comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:47.

[0136] In some embodiments, the genetically modified rodent contains a humanized Il7ra gene in its genome, which encodes a humanized Il7ra protein containing an extracellular domain substantially identical to that of a human IL7RA protein. In some embodiments, the extracellular domain substantially identical to that of a human IL7RA protein exhibits the same functionality (e.g., ligand binding properties) as the extracellular domain of a human IL7RA protein. An extracellular domain or polypeptide that is "substantially identical to the extracellular domain of a human IL7RA protein" can be a polypeptide that is at least 95% identical in sequence to the extracellular domain of a human IL7RA protein, a polypeptide that is at least 98% identical in sequence to the extracellular domain of a human IL7RA protein, or a polypeptide that is at least 99% identical in sequence to the extracellular domain of a human IL7RA protein. An extracellular domain or polypeptide that is "substantially identical to the extracellular domain of a human IL7RA protein" can be a polypeptide that is 100% identical in sequence to the extracellular domain of a human IL7RA protein. Alternatively or additionally, an extracellular domain or polypeptide that is "substantially identical to the extracellular domain of a human IL7RA protein" may be (ii) a polypeptide that differs by no more than 10 amino acids from the extracellular domain of a human IL7RA protein, a polypeptide that differs by no more than 7 amino acids from the extracellular domain of a human IL7RA protein, a polypeptide that differs by no more than 5 amino acids from the extracellular domain of a human IL7RA protein, a polypeptide that differs by no more than 4 amino acids from the extracellular domain of a human IL7RA protein, a polypeptide that differs by no more than 3 amino acids from the extracellular domain of a human TSLPR protein, a polypeptide that differs by no more than 2 amino acids from the extracellular domain of a human TSLPR protein, or a polypeptide that differs by no more than 1 amino acid from the extracellular domain of a human TSLPR protein.Alternatively or additionally, an extracellular domain or polypeptide that is "substantially identical to the extracellular domain of a human IL7RA protein" may be (iii) a polypeptide that differs from the extracellular domain of a human IL7RA protein only at the N-terminal or C-terminal portion of the extracellular domain, for example, by having amino acid additions, deletions, and / or substitutions at the N-terminal and / or C-terminal portion of the extracellular domain (i.e., within 5-10 amino acids from the N- or C-terminus of the extracellular domain). Alternatively or additionally, an extracellular domain or polypeptide that is "substantially identical to the extracellular domain of a human IL7RA protein" may be a polypeptide having one or more of the characteristics detailed in (i) to (iii) above, for example, a polypeptide that is at least 95% identical in sequence to the extracellular domain of a human IL7RA protein and differs from the extracellular domain of a human IL7RA protein by no more than 5 amino acids only in the N-terminal or C-terminal portion of the extracellular domain, or a polypeptide that is at least 98% identical in sequence to the extracellular domain of a human IL7RA protein and differs from the extracellular domain of a human IL7RA protein by no more than 3 amino acids only in the N-terminal or C-terminal portion of the extracellular domain. In a specific embodiment, the human IL7RA protein comprises the amino acid sequence set forth in SEQ ID NO: 43, and the extracellular domain is defined by amino acids 21 to 238 of SEQ ID NO: 43. In some embodiments, the humanized Il7ra gene encodes a humanized Il7ra protein whose extracellular domain is substantially identical to the extracellular domain of the human IL7RA protein set forth in SEQ ID NO:43, i.e., substantially identical to amino acids 21-238 of SEQ ID NO:43. In some embodiments, the humanized Il7ra protein comprises an extracellular domain that includes amino acids 21-238 of SEQ ID NO:43. In some embodiments, the humanized Il7ra protein comprises an extracellular domain that includes amino acids 21-237 of SEQ ID NO:43. In some embodiments, the humanized Il7ra protein comprises an extracellular domain that includes amino acids 21-236 of SEQ ID NO:43. In some embodiments, the humanized Il7ra protein comprises an extracellular domain that includes amino acids 22-238 of SEQ ID NO:43.In some embodiments, the humanized Il7ra protein comprises an extracellular domain comprising amino acids 24-238 of SEQ ID NO:43.

[0137] In some embodiments, the humanized Il7ra gene encodes a humanized Il7ra protein having an extracellular domain that includes the extracellular domain of human IL7RA or a substantial portion thereof, followed by amino acids from the C-terminus of the extracellular domain of rodent Il7ra (e.g., endogenous rodent Il7ra). A "substantial portion of the extracellular domain" of a human IL7RA (or rodent IL7ra) protein refers to a polypeptide that is almost the complete extracellular domain of a human IL7RA (or rodent IL7ra) protein. In some embodiments, a substantial proportion of the extracellular domain includes at least 95% of the full-length extracellular domain sequence. In some embodiments, a substantial proportion of the extracellular domain includes at least 98% of the full-length extracellular domain sequence. In some embodiments, a substantial proportion of the extracellular domain differs from the extracellular domain by the absence of no more than 10 amino acids at the N-terminus or C-terminus of the extracellular domain. In some embodiments, a substantial proportion of the extracellular domain differs from the extracellular domain by the absence of no more than 7 amino acids at the N-terminus or C-terminus of the extracellular domain. In some embodiments, a substantial proportion of the extracellular domain differs from the extracellular domain by the absence of no more than 5 amino acids at the N-terminus or C-terminus of the extracellular domain. In some embodiments, a substantial proportion of the extracellular domain differs from the extracellular domain by the absence of no more than 3 amino acids at the N-terminus or C-terminus of the extracellular domain. For example, the extracellular domain of the human IL7RA protein set forth in SEQ ID NO: 43 is defined by amino acids 21-238, and examples of substantial portions of this extracellular domain can include amino acids 22-238, 23-238, 24-238, 21-237, 21-236, 21-235 of the human IL7RA protein set forth in SEQ ID NO: 43. In some embodiments, a substantial portion of the extracellular domain of human IL7RA includes amino acids 21-236 of SEQ ID NO: 43. In some embodiments, a substantial portion of the extracellular domain of human IL7RA comprises amino acids 21-237 of SEQ ID NO: 43. In some embodiments, a substantial portion of the extracellular domain of human IL7RA comprises amino acids 22-238 of SEQ ID NO: 43. In some embodiments, a substantial portion of the extracellular domain of human IL7RA comprises amino acids 23-238 of SEQ ID NO: 43.In some embodiments, the extracellular domain of a humanized Il7ra protein comprises a substantial portion of the extracellular domain of human IL7RA, followed by 1-3 amino acids from the C-terminus of the extracellular domain of rodent Il7ra (e.g., endogenous rodent Il7ra). In some embodiments, the extracellular domain of a humanized Il7ra protein comprises amino acids 21-236 of SEQ ID NO:43, followed by 2 amino acids from the C-terminus of the extracellular domain of rodent Il7ra. In some embodiments, the humanized Il7ra gene encodes a humanized Il7ra protein containing an extracellular domain comprising amino acids 21-238 of SEQ ID NO: 45 - this extracellular domain comprises amino acids 21-236 of SEQ ID NO: 43 (human IL7RA) and the last two amino acids ("GW") from the C-terminus of the extracellular domain of mouse Il7ra, differing from the extracellular domain of the human IL7RA protein of SEQ ID NO: 43 in two amino acids at the C-terminus of the extracellular domain (with "GW" in humanized Il7ra of SEQ ID NO: 45 as opposed to "EM" in human IL7RA of SEQ ID NO: 43) and otherwise identical to the human IL7RA extracellular domain.

[0138] In some embodiments, the humanized Il7ra gene encodes a humanized Il7ra protein that contains a transmembrane cytoplasmic sequence (i.e., a sequence that includes both a transmembrane domain and a cytoplasmic domain) that is substantially identical to a transmembrane cytoplasmic sequence of a rodent Il7ra protein, such as an endogenous rodent Il7ra protein. In some embodiments, the transmembrane cytoplasmic sequence that is substantially identical to the transmembrane cytoplasmic sequence of an endogenous rodent Il7ra protein exhibits the same functionality (e.g., signal transduction and / or interaction with intracellular molecules) as the transmembrane cytoplasmic sequence of a rodent Il7ra protein, such as an endogenous rodent Il7ra protein. A transmembrane cytoplasmic sequence or polypeptide that is "substantially identical to a transmembrane cytoplasmic sequence of a rodent Tslpr protein" can, in some embodiments, be (i) a polypeptide that is at least 95% identical in sequence to a transmembrane cytoplasmic sequence of a rodent Il7ra protein, or a polypeptide that is at least 98% identical in sequence to a transmembrane cytoplasmic sequence of a rodent Il7ra protein. A transmembrane cytoplasmic sequence or polypeptide that is "substantially identical to a transmembrane cytoplasmic sequence of a rodent Tslpr protein" can be a polypeptide that is identical in sequence to a transmembrane cytoplasmic sequence of a rodent Il7ra protein. Alternatively or additionally, a transmembrane cytoplasmic sequence or polypeptide that is "substantially identical to the transmembrane cytoplasmic sequence of a rodent Tslpr protein" can be (ii) a polypeptide that differs by no more than 5 amino acids from the transmembrane cytoplasmic sequence of a rodent Il7ra protein, a polypeptide that differs by no more than 4 amino acids from the transmembrane cytoplasmic sequence of a rodent Il7ra protein, a polypeptide that differs by no more than 3 amino acids from the transmembrane cytoplasmic sequence of a rodent Il7ra protein, a polypeptide that differs by no more than 2 amino acids from the transmembrane cytoplasmic sequence of a rodent Il7ra protein, or a polypeptide that differs by no more than 1 amino acid from the transmembrane cytoplasmic sequence of a rodent Il7ra protein.Alternatively or additionally, a transmembrane cytoplasmic sequence or polypeptide that is "substantially identical to the transmembrane cytoplasmic sequence of a rodent Tslpr protein" may be (iii) a polypeptide that differs from the transmembrane cytoplasmic sequence of a rodent Il7ra protein only at the N-terminus or C-terminus, for example, by having an amino acid addition, deletion or substitution at the N-terminus or C-terminus portion of the transmembrane cytoplasmic sequence. Alternatively or additionally, a transmembrane cytoplasmic sequence or polypeptide that is "substantially identical to the transmembrane cytoplasmic sequence of a rodent Tslpr protein" may be (iv) a polypeptide that has one or more of the characteristics detailed in (i) to (iii) above, for example, a polypeptide that is at least 95% identical in sequence to the transmembrane cytoplasmic sequence of a rodent Il7ra protein and differs from the transmembrane cytoplasmic sequence of a rodent Il7ra protein only at the N-terminus or C-terminus by no more than 3 amino acids. "N- or C-terminal portion of a transmembrane cytoplasmic sequence" means within 5-10 amino acids from the N-terminus of the transmembrane domain, or within 5-10 amino acids from the C-terminus of the cytoplasmic domain. In some embodiments, a humanized Il7ra protein contains a transmembrane cytoplasmic sequence substantially identical to the transmembrane cytoplasmic sequence of a mouse Il7ra protein (such as an endogenous mouse Il7ra protein) or a transmembrane cytoplasmic sequence of a rat Il7ra protein (such as an endogenous rat Il7ra protein).

[0139] In some embodiments, the humanized Il7ra gene encodes a humanized Il7ra protein that contains a signal peptide substantially identical to that of a rodent Il7ra protein (e.g., an endogenous rodent Il7ra protein). In some embodiments, the humanized Il7ra gene encodes a humanized Il7ra protein that contains a signal peptide substantially identical to that of a human IL7RA protein (e.g., a human IL7RA protein set forth in SEQ ID NO: 43). A signal peptide that is "substantially identical" to a signal peptide of a reference protein (either a human IL7RA protein or a rodent Il7ra protein) can be (i) a polypeptide that is at least 95% identical in sequence to the signal peptide of the reference protein, or a polypeptide that is identical in sequence to the signal peptide of the reference protein. Alternatively or additionally, a signal peptide that is "substantially identical" to a signal peptide of a reference protein can be (ii) a polypeptide that differs from the signal peptide of the reference protein by no more than three amino acids, a polypeptide that differs from the signal peptide of the reference protein by no more than two amino acids, or, in some embodiments, a polypeptide that differs from the signal peptide of the reference protein by no more than one amino acid. Alternatively or additionally, a signal peptide that is "substantially identical" to a signal peptide of a reference protein may be (iii) a polypeptide that differs from the signal peptide of the reference protein only at the N-terminus or C-terminus, for example, by having an amino acid addition, deletion or substitution at the N-terminus or C-terminus portion of the signal peptide, or (iv) a polypeptide that has one or more of the characteristics detailed in (i)-(iii) above, for example, a polypeptide that is at least 95% identical to the signal peptide of the reference protein and differs from the signal peptide of the reference protein only at the N-terminus or C-terminus by no more than 3 amino acids. "N- or C-terminal portion of the signal peptide" means within 5 amino acids from the N- or C-terminus of the signal peptide. In some embodiments, the humanized Il7ra protein comprises a signal peptide that is substantially identical to the signal peptide of a mouse Il7ra protein (such as an endogenous mouse Il7ra protein).In some embodiments, the humanized Il7ra protein comprises a signal peptide substantially identical to that of a rat Il7ra protein (such as an endogenous rat Il7ra protein). In some embodiments, the humanized Il7ra protein comprises a signal peptide substantially identical to that of a human IL7RA protein (e.g., the human IL7RA protein set forth in SEQ ID NO:43).

[0140] In some embodiments, the humanized Il7ra gene in the genome of the genetically modified rodent comprises a nucleotide sequence of a human IL7RA gene ("human IL7RA nucleotide sequence") and a nucleotide sequence of a rodent Il7ra gene ("rodent Il7ra nucleotide sequence", e.g., an endogenous rodent Il7ra nucleotide sequence), wherein the human IL7RA nucleotide sequence encodes at least a substantial portion of the extracellular domain of a human IL7RA protein. In some embodiments, the human IL7RA nucleotide sequence is a cDNA sequence. In some embodiments, the human IL7RA nucleotide sequence in the humanized Il7ra gene encodes the extracellular domain of a human IL7RA protein (e.g., a human IL7RA protein of SEQ ID NO:43). In some embodiments, the human IL7RA nucleotide sequence is a genomic fragment of a human IL7RA gene. In some embodiments, the human IL7RA nucleotide sequence is a genomic fragment of the human IL7RA gene that includes the codon in exon 2 that encodes the first amino acid in the mature protein sequence through exon 5 (i.e., the codon that encodes the amino acid residue that is two amino acids before the start of the transmembrane segment of the human IL7RA protein. In some embodiments, the human IL7RA genomic fragment encodes amino acids 21-238 of the human IL7RA protein (e.g., the human IL7RA protein set forth in SEQ ID NO:43). ... In some embodiments, the human IL7RA genomic fragment encodes amino acids 21-237 of a human IL7RA protein (e.g., a human IL7RA protein set forth in SEQ ID NO:43). In some embodiments, the human IL7RA genomic fragment encodes amino acids 21-236 of a human IL7RA protein (e.g., a human IL7RA protein set forth in SEQ ID NO:43). In some embodiments, the human IL7RA genomic fragment encodes amino acids 21-235 of a human IL7RA protein (e.g., a human IL7RA protein set forth in SEQ ID NO:43). In some embodiments, the human IL7RA genomic fragment encodes amino acids 22-238 of a human IL7RA protein (e.g., a human IL7RA protein set forth in SEQ ID NO:43).In some embodiments, the human IL7RA genomic fragment encodes amino acids 24 to 238 of a human IL7RA protein (e.g., a human IL7RA protein set forth in SEQ ID NO: 43). In some embodiments, the human IL7RA genomic fragment further comprises a 5' portion of intron 5.

[0141] In some embodiments, the humanized Il7ra gene in the genome of the genetically modified rodent comprises a rodent Il7ra nucleotide sequence and a human IL7RA nucleotide sequence, wherein the rodent Il7ra nucleotide sequence encodes a polypeptide substantially identical to the transmembrane cytoplasmic sequence of a rodent Il7ra protein (e.g., an endogenous rodent Il7ra protein). In some embodiments, the rodent Il7ra nucleotide sequence present in the humanized Il7ra gene encodes the transmembrane cytoplasmic sequence of an endogenous rodent Il7ra protein. In some embodiments, the rodent Il7ra nucleotide sequence present in the humanized Il7ra gene is a mouse Il7ra nucleotide sequence, and in some such embodiments, the mouse Il7ra nucleotide sequence comprises a 3' portion of intron 5 and exon 6 through exon 8 of the mouse Il7ra gene (e.g., an endogenous mouse Il7ra gene).

[0142] In some embodiments, the humanized Il7ra gene in the genome of the genetically modified rodent comprises a rodent Il7ra nucleotide sequence upstream (5') of the human IL7RA nucleotide sequence, where the rodent Il7ra nucleotide sequence encodes a polypeptide substantially identical to a signal peptide of a rodent Il7ra protein (e.g., an endogenous rodent Il7ra protein). In some embodiments, the rodent Il7ra nucleotide sequence encoding a polypeptide substantially identical to a signal peptide of a rodent Il7ra protein is a mouse Il7ra nucleotide sequence (e.g., an endogenous mouse Il7ra nucleotide sequence) or a rat Il7ra nucleotide sequence (e.g., an endogenous rat Il7ra nucleotide sequence). In some embodiments, the mouse Il7ra nucleotide sequence in the humanized Il7ra gene includes a portion of exon 1 of the mouse Il7ra gene (e.g., the endogenous mouse Il7ra gene) that encodes the signal peptide of mouse Il7ra, and in some embodiments, the mouse Il7ra nucleotide sequence also includes the 5'UTR of exon 1 of the mouse Il7ra gene.

[0143] In some embodiments, the humanized Il7ra gene is operably linked to a rodent Il7ra 5' regulatory sequence, such as an endogenous rodent Il7ra regulatory sequence, e.g., a 5' transcription regulatory sequence, such as a promoter and / or enhancer, such that expression of the humanized Il7ra gene is under the control of the rodent Il7ra 5' regulatory sequence.

[0144] In some embodiments, the humanized Il7ra gene is at the endogenous rodent Il7ra locus. In some embodiments, the humanized Il7ra gene is at a locus other than the endogenous rodent Il7ra locus, e.g., as a result of random integration. In some embodiments where the humanized Il7ra gene is at a locus other than the endogenous rodent Il7ra locus, the rodent is unable to express rodent Il7ra protein, e.g., as a result of inactivation (e.g., total or partial deletion) of the endogenous rodent Il7ra gene.

[0145] In some embodiments where the humanized Il7ra gene is at the endogenous rodent Il7ra locus, the humanized Il7ra gene results from replacement of the endogenous rodent Il7ra gene at the endogenous rodent Il7ra locus with the nucleotide sequence of the human IL7RA gene.

[0146] In some embodiments, the nucleotide sequence of the endogenous rodent Il7ra gene to be replaced at the endogenous rodent Il7ra locus is a genomic fragment of the endogenous rodent Il7ra gene that encodes at least a substantial portion of the extracellular domain of the rodent Il7ra protein. In some embodiments, the rodent is a mouse, and the mouse Il7ra genomic fragment to be replaced encodes at least a substantial portion of the extracellular domain of the endogenous mouse Il7ra protein. Examples of substantial portions of the extracellular domain of the endogenous mouse Il7ra protein include amino acids 22-238, 23-238, 24-238, 21-237, 21-236, or 21-235 of the endogenous mouse Il7ra protein (e.g., SEQ ID NO: 41). In some embodiments, the mouse Il7ra genomic fragment to be replaced encodes amino acids 21-235 of the endogenous mouse Il7ra protein (e.g., SEQ ID NO: 41). In some embodiments, the mouse Il7ra genomic fragment to be replaced encodes amino acids 21-236 of the endogenous mouse Il7ra protein (e.g., SEQ ID NO: 41). In some embodiments, the mouse Il7ra genomic fragment to be replaced encodes amino acids 21-237 of the endogenous mouse Il7ra protein (e.g., SEQ ID NO: 41). In some embodiments, the mouse Il7ra genomic fragment to be replaced encodes amino acids 21-238 of the endogenous mouse Il7ra protein (e.g., SEQ ID NO: 41). In some embodiments, the mouse Il7ra genomic fragment to be replaced encodes amino acids 22-238 of the endogenous mouse Il7ra protein (e.g., SEQ ID NO: 41) (e.g., SEQ ID NO: 41). In some embodiments, the mouse Il7ra genomic fragment to be replaced encodes amino acids 23-238 of the endogenous mouse Il7ra protein (e.g., SEQ ID NO: 41). In some embodiments, the mouse Il7ra genomic fragment to be replaced includes the codon in exon 1 encoding the first amino acid of the mature Il7ra protein through exon 5 of the mouse Il7ra gene, and in some embodiments, through the 5' portion of intron 5.

[0147] In some embodiments, the nucleotide sequence of the human IL7RA gene that replaces the genomic fragment of the rodent Il7ra gene at the endogenous rodent Il7ra locus is a cDNA sequence. In some embodiments, the human IL7RA nucleotide sequence that replaces the genomic fragment of the rodent Il7ra gene at the endogenous rodent Il7ra locus is a genomic fragment of the human IL7RA gene. In some embodiments, the genomic fragment of the human IL7RA gene that replaces the genomic fragment of the rodent Il7ra gene at the endogenous rodent Il7ra locus comprises all or a portion of an exon of the human IL7RA gene that encodes at least a substantial portion of the extracellular domain of the human IL7RA protein. In some embodiments, the human IL7RA genomic fragment encodes human IL7RA, e.g., amino acids 21-238 of human IL7RA as set forth in SEQ ID NO:43. In some embodiments, the human IL7RA genomic fragment encodes amino acids 21-237 of human IL7RA, e.g., human IL7RA set forth in SEQ ID NO: 43. In some embodiments, the human IL7RA genomic fragment encodes amino acids 21-236 of human IL7RA, e.g., human IL7RA set forth in SEQ ID NO: 43. In some embodiments, the human IL7RA genomic fragment encodes amino acids 21-235 of human IL7RA, e.g., human IL7RA set forth in SEQ ID NO: 43. In some embodiments, the human IL7RA genomic fragment encodes amino acids 22-238 of human IL7RA, e.g., human IL7RA set forth in SEQ ID NO: 43. In some embodiments, the human IL7RA genomic fragment encodes amino acids 24-238 of human IL7RA, e.g., human IL7RA set forth in SEQ ID NO: 43. In some embodiments, the human IL7RA genomic fragment comprises a codon in exon 1 encoding the first amino acid of the mature human IL7RA protein through exon 5, and in some embodiments, through the 5' portion of intron 5, of the human IL7RA gene.

[0148] In some embodiments, the human IL7RA nucleotide sequence inserted into the endogenous rodent Il7ra locus is operably linked to a genomic sequence of a rodent Il7ra gene that encodes a polypeptide substantially identical to the transmembrane cytoplasmic sequence of a rodent Il7ra protein (such as an endogenous rodent, mouse, or rat Il7ra protein). In embodiments where the rodent is a mouse, the genomic sequence of the mouse Il7ra gene, in some embodiments, includes exon 6 through exon 8 of the mouse Il7ra gene (e.g., an endogenous mouse Il7ra gene), and in some embodiments, includes the 3' portion of intron 5 through exon 8 of the mouse Il7ra gene (e.g., an endogenous mouse Il7ra gene).

[0149] In some embodiments, the human IL7RA nucleotide sequence inserted into the endogenous rodent Il7ra locus is operably linked to a genomic sequence of a rodent Il7ra gene that encodes a polypeptide substantially identical to a signal peptide of a rodent Il7ra protein (e.g., an endogenous rodent, mouse, or rat Il7ra protein). In embodiments where the rodent is a mouse, the genomic sequence of the mouse Il7ra gene, in some embodiments, includes a portion of exon 1 of the mouse Il7ra gene (e.g., an endogenous mouse Il7ra gene) that encodes the signal peptide of the mouse Il7ra gene, and in some embodiments, includes the 5' UTR of exon 1 of the mouse Il7ra gene (e.g., an endogenous mouse Il7ra gene) and a portion of exon 1 that encodes the signal peptide of mouse Il7ra.

[0150] In some embodiments, the rodent is a mouse, and a genomic fragment of the endogenous mouse Il7ra gene comprising from the first codon of exon 1 through exon 5 (or in some embodiments, through the 5' portion of intron 5) encoding the first mature Il7ra amino acid has been replaced at the endogenous mouse Il7ra locus with a genomic fragment of a human IL7RA gene comprising from the first codon of exon 1 through exon 5 (or in some embodiments, through the 5' portion of intron 5) encoding the first mature IL7RA amino acid. In some embodiments, the humanized Il7ra gene is formed at the endogenous rodent Il7ra locus and includes the 5'UTR and signal peptide coding portion of exon 1 of the mouse Il7ra gene, the mature amino acid coding portion of exons 1 through 5 of the human IL7RA gene, and exons 6 through 8 of the mouse Il7ra gene; in some such embodiments, intron 5 of the humanized Il7ra gene includes the 5' portion of human intron 5 and the 3' portion of endogenous mouse intron 5.

[0151] In some embodiments, the rodent is a mouse comprising a humanized Il7ra gene at the endogenous mouse Il7ra locus, the humanized Il7ra gene encoding a humanized Il7ra protein comprising a signal peptide at least substantially identical to the signal peptide of a human IL7RA protein, an extracellular domain at least substantially identical to the extracellular domain of a human IL7RA protein, and a transmembrane cytoplasmic domain of an endogenous mouse Il7ra protein. In some embodiments, the humanized Il7ra protein comprises the full-length extracellular domain of a human IL7RA protein. In some embodiments, the extracellular domain of the humanized Il7ra protein comprises (i) nearly the full-length extracellular domain of a human IL7RA protein, except for the C-terminal two amino acids of the extracellular domain of the human IL7RA protein, and (ii) the C-terminal two amino acids of the extracellular domain of the endogenous mouse Il7ra protein. In some embodiments, the rodent is a mouse that contains a humanized Il7ra gene at the endogenous mouse Il7ra locus as described in CN111808882A, which is incorporated by reference herein in its entirety.

[0152] In some embodiments, the rodent provided herein is heterozygous for a humanized Il7ra gene in its genome. In some embodiments, the rodent provided herein is homozygous for a humanized Il7ra gene in its genome.

[0153] In some embodiments, the humanized Il7ra gene results in expression of the encoded humanized Il7ra protein in the rodent. In some embodiments, the humanized Il7ra protein is expressed in cells and tissues where the corresponding rodent Il7ra protein in a control rodent (e.g., a rodent that does not have a humanized Il7ra gene) is normally expressed, e.g., on T lymphocytes.

[0154] In some embodiments, a rodent disclosed herein is unable to express a rodent Il7ra protein, e.g., as a result of inactivation (e.g., deletion, in whole or in part) or replacement (in whole or in part) of an endogenous rodent Il7ra gene.

[0155] Further genetic characteristics In some embodiments, the rodent disclosed herein further comprises a humanized Sirpa gene in its genome. Humanization of rodent Sirpa genes is described, for example, in WO2015 / 042557A1 (Regeneron Pharmaceuticals Inc.) and US2019 / 0373867A1 (Beijing Biocytogen), which are incorporated herein by reference in their entireties.

[0156] In some embodiments, the humanized Sirpa gene encodes a humanized Sirpα protein that comprises all or a portion of the extracellular domain of the human SIRPα protein. In some embodiments, the humanized Sirpa gene encodes a humanized Sirpα protein that comprises the extracellular portion of the human SIRPα protein involved in ligand binding (i.e., binding to CD47). In some embodiments, the humanized Sirpa gene encodes a humanized Sirpα protein that comprises a human SIRPα protein, e.g., amino acid residues 28-362 of the human SIRPα protein set forth in GenBank Accession No. NP_001035111.1. In some embodiments, the humanized Sirpa gene encodes a humanized Sirpα protein that comprises the transmembrane and cytoplasmic domains of a rodent Sirpα protein (e.g., an endogenous rodent Sirpα protein). In some embodiments, the humanized Sirpα gene comprises exons 2, 3, and 4 of the human SIRPα gene. In some embodiments, the humanized Sirpα gene is located at the endogenous rodent Sirpα locus. In some embodiments, the humanized Sirpα gene is formed as a result of replacement of exons 2-4 of an endogenous rodent Sirpα gene at the endogenous rodent Sirpα locus with exons 2-4 of the human SIRPα gene. In some embodiments, the humanized Sirpα gene is located at the endogenous rodent Sirpα locus and comprises exon 1 of the endogenous rodent Sirpα gene, exons 2-4 of the human SIRPα gene, and exons 5-8 of the endogenous rodent Sirpα gene, wherein the humanized Sirpα gene is operably linked to a rodent Sirpα promoter at the endogenous rodent Sirpα locus. In some embodiments, the rodent is heterozygous for the humanized Sirpα gene. In some embodiments, the rodent is homozygous for the humanized Sirpα gene. In some embodiments, a rodent comprising a humanized Sirpα gene expresses a humanized Sirpα protein, such as a protein comprising the extracellular domain of a human SIRPα protein and the transmembrane cytoplasmic domain of a rodent Sirpα protein.In some embodiments, a rodent disclosed herein is incapable of expressing an endogenous rodent Sirpα protein (eg, as a result of a disruption or replacement of the endogenous rodent Sirpα gene).

[0157] In some embodiments, the rodent disclosed herein further comprises a humanized Tpo (thrombopoietin) gene in its genome. Humanization of rodent Tpo genes is described, for example, in U.S. Pat. No. 8,541,646 (Regeneron Pharmaceuticals Inc., Yale University, and Institute for Research in Biomedicine IRB), and Rongvaux et al. (Proc Natl Acad Sci USA. 2011; 108(6):2378-2383), which are incorporated herein by reference in their entireties. In some embodiments, the humanization comprises replacement of the endogenous rodent Tpo gene with a human TPO gene. In some embodiments, the rodent expresses human TPO protein from a humanized Tpo gene. In some embodiments, the rodent is heterozygous for the humanized Tpo gene. In some embodiments, the rodent is homozygous for the humanized Tpo gene. In some embodiments, a rodent comprising a humanized Tpo gene is incapable of expressing an endogenous rodent Tpo protein (eg, as a result of disruption or replacement of the endogenous rodent Tpo gene).

[0158] In some embodiments, the rodent disclosed herein further comprises a humanized GM-CSF / IL-3 locus in its genome, where the endogenous rodent GM-CSF gene is replaced with a human GM-CSF gene and the endogenous rodent IL-3 gene is replaced with a human IL-3 gene. Humanization of rodent GM-CSF / IL-3 locus is described, for example, in U.S. Pat. No. 8,541,646 (Regeneron Pharmaceuticals Inc., Yale University, and Institute for Research in Biomedicine IRB) and Willinger et al. (PNAS, 108(6):2390-2395, 2011), both of which are incorporated herein in their entirety. In some embodiments, the rodent is heterozygous for the humanized GM-CSF / IL-3 locus. In some embodiments, the rodent is homozygous for the humanized GM-CSF / IL-3 locus. In some embodiments, the rodent expresses human GM-CSF and human IL-3 from a humanized GM-CSF / IL-3 locus. In some embodiments, the rodent disclosed herein is incapable of expressing endogenous rodent GM-CSF protein and incapable of expressing endogenous rodent IL-3 protein (e.g., as a result of disruption or replacement of the endogenous rodent GM-CSF / IL-3 locus).

[0159] In some embodiments, a rodent disclosed herein has its endogenous RAG2 gene disrupted, and in some embodiments, the rodent is homozygous for the disruption (RAG2- / - or RAG knockout) and is unable to express endogenous RAG2 protein. In some embodiments, a rodent disclosed herein has its endogenous IL-2RG gene disrupted, and in some embodiments, the rodent is homozygous for the disruption (IL-2RG- / or IL-2RG knockout) and is unable to express endogenous IL-2RG protein (also known as "γc"). RAG2 and IL-2RG double knockout (DKO) rodents are known immunodeficient rodents (see, e.g., Traggiai E et al. (2004) Development of a human adaptive immune system in cord blood cell-transplanted mice, Science 304:104-107, incorporated herein by reference in its entirety) and are readily available commercially (e.g., from Taconic Biosciences, Inc., New York).

[0160] In some embodiments, a rodent disclosed herein comprises in its genome a humanized Tslp gene and a humanized Sirpα gene, and is homozygous null for both the RAG2 gene and the IL-2RG gene. In some such embodiments, the rodent further comprises in its genome a humanized Tpo gene, and / or a humanized GM-CSF / IL-3 locus. The rodent can be heterozygous or homozygous for the humanized genes.

[0161] In some embodiments, a rodent disclosed herein comprises in its genome a humanized Tslp gene, a humanized Tslpr gene, and a humanized Sirpα gene, and is homozygous null for both the RAG2 gene and the IL-2RG gene. In some such embodiments, the rodent further comprises in its genome a humanized Tpo gene and / or a humanized GM-CSF / IL-3 locus. The rodent can be homozygous or heterozygous for the humanized genes.

[0162] In some embodiments, a rodent disclosed herein comprises in its genome a humanized Tslp gene, a humanized Tslpr gene, a humanized Il7ra gene, and a humanized Sirpα gene, and is homozygous null for both the RAG2 gene and the IL-2RG gene. In some such embodiments, the rodent further comprises in its genome a humanized Tpo gene, and / or a humanized GM-CSF / IL-3 locus. The rodent can be homozygous or heterozygous for the humanized genes.

[0163] Rodent species and strains In some embodiments, rodents of the present disclosure include, by way of non-limiting example, mice, rats, and hamsters, hi some embodiments, the rodent is selected from the superfamily Muroidea. In some embodiments, a rodent of the present disclosure is an animal from a family selected from Calomyscidae (e.g., kangaroo hamsters), Cricetidae (e.g., hamsters, New World rats and mice, and voles), Muridae (pure-breed mice and rats, gerbils, spiny mice, and maned mice), Nesomyidae (climbing mice, rock mice, with-tailed rats, Madagascar rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rats, bamboo rats, and zokors). In some embodiments, the rodent of the present disclosure is selected from a purebred mouse or rat (Muridae), a gerbil, a spiny mouse, and a maned mouse. In some embodiments, the mouse of the present disclosure is from a member of the Muridae family.

[0164] In some embodiments, the rodent is a mouse. In some embodiments, the rodent is a mouse of a C57BL strain selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the rodent is a 129 strain of mouse selected from the group consisting of strains that are 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129 / SvJae, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al., 1999, Mammalian Genome 10:836; Auerbach et al., 2000, Biotechniques 29(5):1024-1028, 1030, 1032). In some embodiments, the rodent is a mouse that is a mix of 129 and C57BL / 6 strains. In some embodiments, the rodent is a mouse that is a mix of the aforementioned 129 strains, or a mix of the aforementioned BL / 6 strains. In some embodiments, the rodent is a mouse of a BALB strain, e.g., a BALB / c strain. In some embodiments, the rodent is a mouse that is a mix of a BALB strain and another aforementioned strain.

[0165] In some embodiments, the rodent is a rat. In some specific embodiments, the rat is selected from Wistar rats, LEA strains, Sprague Dawley strains, Fischer strains, F344, F6, and Dark Agouti. In some embodiments, the rat strains described herein are a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0166] Genetically modified rodent tissues and cells In some embodiments, disclosed herein is an isolated rodent cell or tissue whose genome comprises a humanized Tslp gene, a humanized Tslpr gene, a humanized Il7ra gene, or a combination thereof. In some embodiments, the isolated rodent cell or tissue further comprises one or more of the additional genetic modifications described above (e.g., a humanized Sirpa gene, a RAG2- / - and an IL-2RG- / -, a humanized Tpo gene, or a humanized GM-CSF / IL-3 locus).

[0167] In some embodiments, the tissue is selected from fat, bladder, brain, breast, bone marrow, eye, heart, intestine, kidney, liver, lung, lymph node, muscle, pancreas, plasma, serum, skin, spleen, stomach, thymus, testis, egg, and combinations thereof.

[0168] In some embodiments, the cell is selected from an epithelial cell, a kerotinocyte, a dendritic cell, a lymphocyte (e.g., a B cell or a T cell), a macrophage, a mast cell, and a basophil. In some embodiments, the isolated rodent cell is a rodent embryonic stem cell. In some embodiments, the isolated rodent cell is a rodent egg or a rodent sperm.

[0169] Compositions and methods for producing humanized rodents Disclosed herein are targeting vectors (or nucleic acid constructs) that contain a human TSLP nucleotide sequence, a human TSLPR nucleotide sequence, or a human IL7RA nucleotide sequence that is desirably integrated into a rodent locus to form a humanized gene as described herein.

[0170] In some embodiments, the targeting vector comprises a human TSLP nucleotide sequence that encodes at least a substantial portion of the mature protein sequence of the human TSLP protein described herein above. In some embodiments, the human TSLP nucleotide sequence encodes a polypeptide comprising amino acids 29-159 of SEQ ID NO: 3. In some embodiments, the human TSLP nucleic acid sequence comprises exon 1, which begins with the codon encoding the first amino acid of the mature protein sequence, through the stop codon in exon 4 of the human TSLP gene.

[0171] In some embodiments, the targeting vector comprises a human TSLPR nucleotide sequence encoding at least a substantial portion of the extracellular domain of a human TSLPR protein described herein. In some embodiments, the human TSLPR nucleotide sequence encodes a polypeptide comprising amino acids 27-231 of SEQ ID NO: 23. In some embodiments, the human TSLPR nucleotide sequence comprises exon 2 through the codon of exon 6 that encodes the last extracellular domain amino acid of the human TSLPR gene.

[0172] In some embodiments, the targeting vector comprises a human IL7RA nucleotide sequence encoding at least a substantial portion of the extracellular domain of the human IL7RA protein described herein above. In some embodiments, the human IL7RA nucleotide sequence encodes a polypeptide comprising amino acids 21-236 of SEQ ID NO: 43. In some embodiments, the human IL7RA nucleotide sequence comprises from the codon in exon 1 encoding the first amino acid of the mature IL7RA protein through exon 5 (and in some embodiments, through the 5' portion of intron 5) of the human IL7RA gene.

[0173] The targeting vector also comprises 5' and 3' rodent sequences flanking the integrated human nucleotide sequence, also known as 5' and 3' homology arms, which mediate homologous recombination and integration of the human nucleotide sequence into a target rodent locus (e.g., an endogenous rodent Tslp locus, an endogenous rodent Tslpr locus, or an endogenous rodent Il7ra locus) to form a humanized gene as described herein above. Typically, the 5' and 3' flanking rodent sequences are nucleotide sequences that flank the corresponding rodent nucleotide sequence at the target rodent locus that is to be replaced by the human nucleotide sequence. In some embodiments, the targeting vector comprises a humanized gene as described herein above. In some embodiments, the targeting vector comprises a humanized Tslp gene comprising a human TSLP nucleotide sequence and a rodent Tslp nucleotide sequence as described herein above. In some embodiments, the targeting vector comprises a humanized Tslpr gene comprising the human TSLPR nucleotide sequence and the rodent Tslpa nucleotide sequence described hereinabove. In some embodiments, the targeting vector comprises a humanized Il7ra gene comprising the human IL7RA nucleotide sequence and the rodent Il7ra nucleotide sequence described hereinabove.

[0174] In some embodiments, the targeting vector comprises a selection marker gene. The selection marker gene can be inserted into an intron of the human genomic sequence to be integrated. In some embodiments, the selection marker gene is provided as an autodeletion cassette that can be deleted after successful integration of the human nucleotide sequence.

[0175] In an exemplary embodiment, the targeting vector is generated from a bacterial artificial chromosome (BAC) clone carrying rodent Tslp, Tslpr, or Il7ra genomic DNA using bacterial homologous recombination and VELOCIGENE® technology (see, e.g., US 6,586,251 and Valenzuela et al. (2003) Nature Biotech. 21(6):652-659, which are incorporated by reference in their entirety). As a result of bacterial homologous recombination, the rodent genomic sequence is deleted from the BAC clone and the human nucleotide sequence is inserted, resulting in a modified BAC clone that carries the human nucleotide sequence flanked by 5' and 3' rodent homology arms. In some embodiments, the human nucleotide sequence can be a cDNA sequence or human genomic DNA. Once linearized, the modified BAC clone can be introduced into rodent embryonic stem (ES) cells.

[0176] In some embodiments, the present invention provides for the use of the targeting vectors described herein to generate modified rodent embryonic stem (ES) cells. The targeting vectors can be introduced into rodent ES cells, for example, by electroporation. Both mouse and rat ES cells have been described in the art. See, for example, US 7,576,259, US 7,659,442, US 7,294,754, and US2008-0078000 A1 (all of which are incorporated by reference in their entireties), which describe mouse ES cells and the VELOCIMOUSE® method of generating genetically modified mice, US 2014 / 0235933 A1 (Regeneron Pharmaceuticals, Inc.), US 2014 / 0310828 A1 (Regeneron Pharmaceuticals, Inc.), which describe methods of generating rat ES cells and genetically modified rats, Tong et al. (2010) Nature 467:211-215, and Tong et al. (2011) Nat Protoc.6(6):doi:10.1038 / nprot.2011.338, all of which are incorporated by reference in their entireties, which can be used to generate modified rodent embryos, which can then be used to generate rodent animals.

[0177] In some embodiments, ES cells can be selected that have a desired human nucleotide sequence (e.g., human TSLP, human TSLPR, or human IL7RA nucleotide sequence) integrated into their genome. In some embodiments, the ES cells are selected based on rodent allele loss and / or human allele gain assays. In some embodiments, the selected ES cells are then used as donor ES cells for injection into pre-morula stage embryos (e.g., 8-cell stage embryos) using the VELOCIMOUSE® method (see, e.g., US 7,576,259, US 7,659,442, US 7,294,754, and US2008-0078000 A1, all of which are incorporated by reference in their entireties), or the methods described in US 2014 / 0235933 A1 and US2014 / 0310828 A1, both of which are incorporated by reference in their entireties. In some embodiments, embryos containing the donor ES cells are incubated and implanted into surrogate mothers to produce F0 rodents. Rodent offspring carrying human nucleotide sequences can be identified by genotyping DNA isolated from tail fragments using rodent allele loss and / or human allele gain assays.

[0178] In some embodiments, rodents heterozygous for the humanized gene can be crossed to produce homozygous rodents.

[0179] The humanized rodents described herein (i.e., rodents comprising a humanized Tslp gene, a humanized Tslpr gene, a humanized Il7ra gene, or a combination thereof) can be bred or crossed with another rodent. Thus, methods of breeding as well as the progeny resulting from such breeding are also embodiments of the present disclosure.

[0180] In some embodiments, a method is provided that includes mating a first rodent as described herein above, for example a rodent whose genome comprises a humanized Tslp gene, a humanized Tslpr gene, a humanized Il7ra gene, or a combination thereof, with a second rodent to obtain a progeny rodent whose genome comprises a humanized Tslp gene, a humanized Tslpr gene, and / or a humanized Il7ra gene. The progeny may have other desirable phenotypes or genetic modifications inherited from the second rodent used in the mating. In some embodiments, the progeny rodent is heterozygous for the humanized gene(s) from the first rodent. In some embodiments, the progeny rodent is homozygous for the humanized gene(s) from the first rodent. In some embodiments, the second rodent used in breeding contains one or more of additional genetic modifications, such as a humanized Sirpa gene, RAG2- / - and IL-2RG- / -, a humanized Tpo gene, or a humanized GM-CSF / IL-3 locus.

[0181] In some embodiments, a progeny rodent is provided whose genome comprises a humanized Tslp gene, a humanized Tslpr gene, a humanized Il7ra gene, or a combination thereof, the progeny rodent being produced by a method comprising mating a first rodent whose genome comprises a humanized gene or genes with a second rodent. In some embodiments, the progeny rodent is heterozygous for the humanized gene(s) from the first rodent. In some embodiments, the progeny rodent is homozygous for the humanized gene(s) from the first rodent.

[0182] In some embodiments, an in vitro method for generating a genetically modified rodent cell is disclosed herein, comprising introducing into a rodent cell a targeting vector comprising a human TSLP nucleic acid sequence encoding at least a substantial portion of the mature protein sequence of the human TSLP protein flanked by rodent homology arms that mediate integration of the human TSLP nucleotide sequence into the endogenous rodent Tslp locus, resulting in replacement of the rodent Tslp genomic DNA with the human TSLP nucleic acid sequence to form a humanized Tslp gene as described herein, thereby generating a genetically modified rodent cell. In some embodiments, the rodent cell is a mouse cell or a rat cell. In some embodiments, the rodent cell is a rodent ES cell, and the method generates a genetically modified rodent ES cell.

[0183] In some embodiments, an in vitro method for generating genetically modified rodent cells is disclosed herein, comprising introducing into rodent cells a targeting vector comprising a human TSLPR nucleic acid sequence encoding at least a substantial portion of the extracellular domain of the human TSLPR protein, flanked by rodent homology arms that mediate the integration of the human TSLPR nucleotide sequence into endogenous rodent Tslpr locus, resulting in replacement of rodent Tslpr genomic DNA with the human TSLPR nucleic acid sequence, forming the humanized Tslpr gene described herein, thereby generating genetically modified rodent cells. In some embodiments, the rodent cells are mouse or rat cells. In some embodiments, the rodent cells are rodent ES cells, and the method generates genetically modified rodent ES cells.

[0184] In some embodiments, an in vitro method for generating a genetically modified rodent cell is disclosed herein, comprising introducing into a rodent cell a targeting vector comprising a human IL7RA nucleic acid sequence encoding at least a substantial portion of the extracellular domain of the human IL7RA protein flanked by rodent homology arms that mediate integration of the human IL7RA nucleotide sequence into an endogenous rodent Il7ra locus, resulting in replacement of the rodent Il7ra genomic DNA with the human IL7RA nucleic acid sequence to form a humanized Il7ra gene as described herein, thereby generating a genetically modified rodent cell. In some embodiments, the rodent cell is a mouse cell or a rat cell. In some embodiments, the rodent cell is a rodent ES cell, and the method generates a genetically modified rodent ES cell.

[0185] Methods using humanized rodents The rodents disclosed herein provide a useful in vivo system and source of biomaterials for identifying and testing potential compounds for the treatment of human diseases, including, inter alia, diseases associated with TSLP signaling, such as Th2-driven allergic diseases, asthma, and cancer.

[0186] In some embodiments, the rodents disclosed herein are used to develop agents that target TSLP signaling, e.g., via targeting human TSLP, human TSLPR, or human IL7RA. In some embodiments, the rodents disclosed herein are used to screen and develop candidate agents (e.g., antibodies) that specifically bind to human TSLP, human TSLPR, or human IL7RA. In some embodiments, the rodents disclosed herein are used to determine the binding profile of an agent (e.g., an antibody). In some embodiments, the rodents disclosed herein are used to measure the effect of blocking or modulating human TSLP, human TSLPR, or human IL7RA activity. In some embodiments, the rodents disclosed herein are exposed to candidate agents that bind to and inhibit human TSLP and analyzed for effects on human TSLP-dependent processes.

[0187] In some embodiments, the genetically modified rodents described herein are used as models of allergic disease. In some embodiments, the allergic disease includes airway inflammation (e.g., asthma).

[0188] In some embodiments, the ova-alum model of lung inflammation is used to evaluate Tslp signaling. The ova-alum model of lung inflammation is well documented in the art (Al-Shami et al., JEM Vol. 202, No. 6, 829-839, 2005; Chu et al., J. Allergy Clin Immunol 2013; 131: 187-200, which are incorporated herein by reference in their entirety). In some embodiments, OVA emulsified in aluminum hydroxide, or aluminum hydroxide alone (as a control), is administered intraperitoneally to rodents (e.g., mice, such as mice humanized for one or more of Tslp, Tslpr, and / or Il7ra disclosed herein, or non-humanized wild-type mice). Mice are then challenged intranasally with OVA and subsequently analyzed for parameters indicative of pulmonary inflammation, including, for example, serum ova-specific IgE and ova-specific-IgG1, goblet cell metaplasia, and / or lung tissue eosinophilia. In some embodiments, pulmonary expression of Muc5ac, a representative mucin gene overexpressed in the airways of asthmatic lungs, can be analyzed after challenge and serve as a surrogate endpoint for goblet cell metaplasia. In an exemplary protocol, 50 μg of OVA emulsified in 2 mg of aluminum hydroxide, or 2 mg of aluminum hydroxide alone, is administered intraperitoneally to rodents (e.g., mice such as mice doubly humanized for Tslp and Tslpr, mice triple humanized for Tslp, Tslpr, and Il7ra, or non-humanized wild-type mice, on days 1 and 14). Anesthetized mice are challenged intranasally with 150 μg of OVA in PBS for 4 days, starting on day 21. Mice are analyzed for serum Ova-specific IgE and Ova-specific-IgG1, goblet cell metaplasia, and / or lung tissue eosinophilia 24 hours after the last challenge. In some embodiments, pulmonary expression of Muc5ac, a representative mucin gene overexpressed in the airways of asthmatic lungs, can be analyzed after challenge and serve as a surrogate endpoint for goblet cell metaplasia.

[0189] In some embodiments, airway inflammation may be induced in rodents by intranasal administration of an allergen (e.g., house dust mite extract or "HDM" model) at one or more doses over a period of time, and airway inflammation may be measured based on mucus accumulation, eosinophil infiltrating cells in bronchoalveolar lavage fluid, levels of total circulating IgE, and / or changes in expression profiles measurable by microarray expression analysis. The efficacy of a candidate therapeutic agent may be determined by measuring whether the degree of airway inflammation is reduced as a result of administration of the agent in either the ova-alum model or the HDM model. The allergen used to induce airway inflammation and the agent being tested may be administered at the same time or at different times. In some embodiments, the allergen is given to the rodent in one or more doses, and the agent being tested is administered to the rodent after at least one allergen challenge has been administered to the rodent.

[0190] In some embodiments, the allergic disease includes skin inflammation or atopic dermatitis. Skin inflammation can be induced in rodents by damaging the skin and exposing the damaged skin to an allergen (e.g., bacterial toxins or dust mite extracts) for a period of time and at one or more doses. The efficacy of the drug can be determined by measuring whether skin inflammation (e.g., as determined by assessing IgE levels, pruritus, epidermal thickening, and other typical symptoms of atopic dermatitis) is reduced as a result of administration of the drug.

[0191] In some embodiments, the rodents disclosed herein are used as animal models of cancer, such as Th2-driven cancers, for example, to evaluate the efficacy of therapeutic agents targeting human cancer cells. In various embodiments, the rodents disclosed herein are implanted with human cancer cells, and drug candidates targeting such human cancer cells are administered to the rodent. The therapeutic efficacy of the agent is then determined by monitoring the human cancer cells in the rodent after administration of the agent, for example, by assessing whether the growth or metastasis of the human cancer cells in the rodent is inhibited as a result of administration of the agent. Human cancer cells suitable for implantation into rodents include, for example, breast cancer cells, lung cancer cells, pancreatic cancer cells, colon cancer cells, melanoma, among others. Agents that can be tested in non-human animals include both small molecule compounds, i.e., compounds with a molecular weight of less than 1500 kD, 1200 kD, 1000 kD, or 800 daltons, and large molecule compounds (such as proteins, e.g., antibodies) that are intended to have a therapeutic effect on the treatment of human diseases and conditions by targeting (e.g., binding to and / or acting on) human cells.

[0192] This description is further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, and published patent applications cited throughout this application) are expressly incorporated herein by reference in their entireties. EXAMPLES

[0193] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods claimed herein are made and evaluated, and are intended to be merely illustrative and are not intended to limit the disclosure.

[0194] Example 1 Generation of humanized Tslp mice The mouse Tslp locus was humanized using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome couple with high-resolution expression analysis. Nat. Biotech. 21(6):652-659, both of which are incorporated herein by reference). The resulting humanized Tslp locus included the mouse Tslp promoter, mouse Tslp exon 1, a portion of mouse Tslp exon 2 (from the 5' end of exon 2 to the codon encoding the last amino acid of the mouse Tslp signal peptide), a portion of human TSLP exon 1 (from the codon encoding the first amino acid of the mature human TSLP protein to the 3' end of exon 1), to the stop codon in human TSLP exon 4, followed by the mouse Tslp 3' UTR and downstream mouse genomic sequence. See Figures 1A-1C.

[0195] To humanize the mouse Tslp locus, a targeting nucleic acid construct was generated based on the following mouse and human sequences: [Table 4] [Table 5-1] [Table 5-2]

[0196] The targeting nucleic acid construct contained the following from 5' to 3': (i) a 5' mouse homology arm 114.3 kb upstream of the codon in mouse Tslp exon 2, which encodes the first amino acid of the mature mouse Tslp protein (see FIG. 1B ); (ii) 1.9 kb of human TSLP genomic sequence, designated as "Human Genomic Fragment 1," beginning with the codon in human TSLP exon 1 encoding the first amino acid of the mature human TSLP protein (amino acid 29) and ending 257 bp after the end of human TSLP exon 3, see FIG. 1B; (iii) a 4.4 kb selection cassette designated as "Floxed HUb-Puro" (a puromycin resistance gene operably linked to the human ubiquitin promoter, flanked by LoxP sites) inserted into intron 3 of human TSLP, see FIG. 1B ; (iv) 2.2 kb of human TSLP genomic sequence, designated "Human Genomic Fragment 2," beginning 258 bp after the end of human TSLP exon 3 and ending at the stop codon in human TSLP exon 4, see FIG. 1B; and (v) A 3' mouse homology arm of approximately 65.3 kb containing the 3' UTR sequence of mouse Tslp exon 5 and downstream mouse genomic sequences, see FIG. 1B.

[0197] The targeting nucleic acid construct was electroporated into F1H4 mouse embryonic stem (ES) cells. Successful integration was confirmed by modification of alleles (MOA) assay, for example, as described in Valenzuela et al., supra. The primers and probes used in the MOA assay to detect the presence of the human TSLP sequence and to confirm the loss and / or retention of the mouse Tslp sequence are shown in Table 6, and their positions are shown in Figure 1B. [Table 6-1] [Table 6-2]

[0198] After selecting for properly targeted ES cell clones, the puromycin selection cassette was excised. The coding sequence and encoded amino acid sequence of the humanized Tslp gene are set forth in SEQ ID NO:6 and SEQ ID NO:5, respectively. An alignment of mouse Tslp (SEQ ID NO:1), human TSLP (SEQ ID NO:3), and humanized Tslp (SEQ ID NO:5) protein sequences is provided in FIG. IF.

[0199] The positively targeted ES cells were used as donor ES cells and microinjected into mouse embryos at the pre-morula (8-cell) stage by the VELOCIMOUSE® method (see, e.g., US 7,576,259, US 7,659,442, US 7,294,754, and US2008-0078000 A1, all of which are incorporated herein by reference in their entirety). Mouse embryos containing donor ES cells were incubated in vitro and then implanted into surrogate mothers to generate F0 mice entirely derived from donor ES cells. Mice carrying the humanized Tslp gene were identified by genotyping using the MOA assay described above. Mice heterozygous for the humanized Tslp gene were bred until homozygous.

[0200] To determine whether mice homozygous for Tslp humanization expressed humanized Tslp protein, mice were euthanized and blood was drawn via cardiac puncture. Blood was collected in serum separator tubes and serum was prepared. Human TSLP levels in serum were determined using the Human Quantikine TSLP ELISA (R&D Systems, Cat. No. DTSLP0) according to the manufacturer's instructions. To verify the species specificity of the ELISA, 1000 pg / mL of recombinant mouse Tslp (R&D Systems Cat. No. 555-TS-010) was also used as a negative control (data not shown) and normal human serum (NHS) was used as a positive control. Mice heterozygous for the Tslp humanization described above were found to express mature human TSLP in serum (Figure 1G).

[0201] Example 2. Generation of humanized Tslpr mice The mouse Tslpr locus was humanized using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome couple with high-resolution expression analysis. Nat. Biotech. 21(6):652-659, both of which are incorporated herein by reference). The resulting humanized Tslpr locus contained the mouse Tslpr promoter, mouse Tslpr exon 1 (including the 5'UTR, and sequences encoding the mouse Tslpr signal peptide and the first 7 amino acids of the mouse Tslpr mature protein), part of mouse Tslpr intron 1 (up to 328 bp before exon 2), part of human TSLPR intron 1 (starting 909 bp before exon 2), human TSLPR exon 2 through the first 47 bp of exon 6 (encoding essentially the human TSLPR extracellular domain, i.e., from amino acid 27 to just before the transmembrane domain), mouse Tslpr exon 6 starting at bp 48 through exon 8 (encoding the mouse Tslpr transmembrane and intracellular domains and including the mouse Tslpr 3'UTR), followed by downstream mouse genomic sequence. See Figures 2A-C.

[0207] To humanize the mouse Tslpr locus, a targeting nucleic acid construct was generated based on the following sequence information: [Table 7] [Table 8]

[0202] The targeting nucleic acid construct contained the following from 5' to 3': (i) the 5′ mouse homology arm from approximately 29.1 kb up to 328 bp before mouse Tslpr exon 2, see FIG. 2B ; (ii) an autodeletion selection cassette containing a neomycin resistance gene operably linked to the human ubiquitin promoter flanked by LoxP sites ("Floxed HUb-Neo"), see FIG. 2B; (iii) a 13,743 bp nucleic acid sequence of human TSLPR, beginning 909 bp before exon 2 of human TSLPR and encoding substantially the human TSLPR extracellular domain, i.e., amino acid 27 to amino acid 231 (in human TSLPR: amino acids 1 to 22 constituting the signal peptide, and amino acids 232 to 252 constituting the transmembrane domain), through the first 47 bp of exon 6, see FIG. 2B ; and (iv) a 3′ mouse homology arm of approximately 133.2 kb beginning at bp 48 of exon 6 and extending to exon 8, encoding the mouse Tslpr transmembrane and intracellular domains and including the mouse Tslpr 3′ UTR followed by downstream mouse genomic sequences; see FIG. 2B .

[0203] The targeting nucleic acid construct was electroporated into mouse embryonic stem (ES) cells. Successful integration was confirmed by modification of allele (MOA) assay, for example as described in Valenzuela et al., supra. The primers and probes used in the MOA assay to detect the presence of the human TSLPR sequence and to confirm the loss and / or retention of the mouse Tslpr sequence are shown in Table 9, and their positions are shown in FIG. 2B. After selecting the properly targeted ES cell clones, the neomycin selection cassette can be excised. The genomic sequences of the targeted (humanized) Tslpr allele with and without the cassette are listed in SEQ ID NO:63 and 64, respectively. The coding sequence and the encoded amino acid sequence of the humanized Tslpr gene are listed in SEQ ID NO:26 and SEQ ID NO:25, respectively. An alignment of the mouse Tslpr (SEQ ID NO:21), human TSLPR (SEQ ID NO:23), and humanized Tslpr (SEQ ID NO:25) protein sequences is provided in FIG. 2F. [Table 9-1] [Table 9-2]

[0204] The positively targeted ES cells were used as donor ES cells and microinjected into mouse embryos at the pre-morula (8-cell) stage by the VELOCIMOUSE® method (see, e.g., US 7,576,259, US 7,659,442, US 7,294,754, and US2008-0078000 A1, all of which are incorporated herein by reference in their entirety). Mouse embryos containing donor ES cells were incubated in vitro and then implanted into surrogate mothers to generate F0 mice entirely derived from donor ES cells. Mice carrying the humanized Tslpr gene were identified by genotyping using the MOA assay described above. Mice heterozygous for the humanized Tslpr gene were bred until homozygous.

[0205] Example 3. Generation of humanized Il7ra mice The mouse Il7ra locus was humanized using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome couple with high-resolution expression analysis. Nat. Biotech. 21(6):652-659, both of which are incorporated by reference in their entireties). The resulting humanized Il7ra locus contained the mouse Il7ra promoter, part of mouse Il7ra exon 1 (including the 5'UTR and the first 68 bp starting from the start codon, encoding the mouse Il7ra signal peptide and the first 3 amino acids of the mouse Il7ra mature protein), part of human IL7RA exon 1 (the last 14 bp of exon 1), human IL7RA intron 1, human IL7RA exon 2 to exon 5, part of human IL7RA intron 5, part of mouse Il7ra intron 5, mouse Il7ra exon 6 to exon 8 (encoding the last two amino acids of the mouse Il7ra extracellular domain, the transmembrane and intracellular domains of mouse Il7ra, and including the mouse Il7ra 3'UTR). See Figures 3A-3F.

[0206] To humanize the mouse Il7ra locus, a targeting nucleic acid construct was generated based on the following sequence information: [Table 10] [Table 11]

[0207] The targeting nucleic acid construct contained the following from 5' to 3': (i) the 5' mouse homology arm of approximately 48.8 kb (from the mouse Il7ra 5' sequence to part of exon 1, i.e., the 5' UTR and the first 68 bp starting from the start codon), see Figure 3B and Figure 3E; (ii) 126 bp human genomic fragment 1 (SEQ ID NO:69), which includes the last 14 bp of exon 1 and the first 112 bp of intron 1 of human IL7RA; see Figures 3B and 3E; (iii) an approximately 5.2 kb autodeletion selection cassette containing a hygromycin resistance gene operably linked to the human ubiquitin promoter flanked by LoxP sites ("Floxed HUb-Hyg"), see FIG. 3B; (iv) human genomic fragment 2 of 17,106 bp containing the 3' portion of intron 1, exon 2 to exon 5, and the 5' portion of intron 5 of human IL7RA (see FIG. 3B ); and (v) The 3' portion of intron 5 of mouse Il7ra, from exon 6 to exon 8 (including the mouse Il7ra 3' UTR), followed by downstream mouse genomic sequences, approximately 124.3 kb of 3' mouse homology, see Figures 3B and 3F. Exons 6 to 8 of mouse Il7ra encode the last two amino acids (Gly-Trp) of the extracellular domain, the transmembrane domain, and the intracellular domain of mouse Il7ra.

[0208] The targeting nucleic acid construct was electroporated into F1H4 mouse embryonic stem (ES) cells. Successful integration was confirmed by modification of allele (MOA) assay, e.g., as described in Valenzuela et al., supra. Primers and probes used in the MOA assay to detect the presence of human IL7RA sequence and to confirm loss and / or retention of mouse Il7ra sequence are listed in Table 12 and their positions are shown in Figure 3B. After selecting properly targeted ES cell clones, the hygromycin selection cassette can be excised. The genomic sequences of the targeted (humanized) Il7ra allele with and without the cassette are listed in SEQ ID NOs: 65 and 66, respectively. The coding sequence and encoded amino acid sequence of the humanized Il7ra gene are listed in SEQ ID NOs: 46 and 45, respectively. An alignment of mouse Il7ra (SEQ ID NO: 41) and human IL7RA (SEQ ID NO: 43) protein sequences is provided in Figure 3F. [Table 12]

[0209] The positively targeted ES cells were used as donor ES cells and microinjected into mouse embryos at the pre-morula (8-cell) stage by the VELOCIMOUSE® method (see, e.g., US 7,576,259, US 7,659,442, US 7,294,754, and US2008-0078000 A1, all of which are incorporated herein by reference in their entirety). Mouse embryos containing donor ES cells were incubated in vitro and then implanted into surrogate mothers to generate F0 mice entirely derived from donor ES cells. Mice carrying the humanized Il7ra gene were identified by genotyping using the MOA assay described above. Mice heterozygous for the humanized Il7ra gene were bred to homozygosity.

[0210] Example 4. Wild-type, double humanized mice (Tslp hu / hu / Tslpr hu / hu ) and triple humanized mice (Tslp hu / hu / Tslpr hu / hu / Il7ra hu / hu Ova-alum-induced type 2 driven inflammation in mice To confirm that the various humanized mouse strains have comparable pathology in models of type-2 driven inflammation, an ovalbumin (OVA) / alum-induced lung inflammation model was employed in which a role for TSLP has been previously reported (Chu, et al., J Allergy Clin Immunol 2013;131:187-200.e1-8, incorporated herein by reference in its entirety). Traditional type-2 inflammation endpoints, such as circulating levels of antigen-specific IgE and IgG1, lung tissue eosinophil infiltration and pulmonary expression of Muc5ac, a representative mucin gene overexpressed in the airways of asthmatic lungs, as well as a surrogate endpoint of goblet cell metaplasia (GCM) (Wills-Karp, et al., Science 1998;282:2258-61, incorporated herein by reference in its entirety), were measured in three mouse strains: wild-type mice (WT), double humanized mice (TSLP), and pulmonary eosinophilic infiltration (PIE). hu / hu / Tslpr hu / hu ), and triple humanized mice (Tslp hu / hu / Tslpr hu / hu / IL7R hu / hu The humanization of each of the Tslp molecule, the Tslpr molecule, and the IL7R molecule was as described in Examples 1, 2, and 3, respectively.

[0211] Because the lung is a highly vascularized organ, prior to gating on eosinophils, cells infiltrating the lung (lung tissue) were distinguished from cells circulating in the pulmonary vasculature (pulmonary circulation) using a CD45-based intravascular labeling technique (Anderson, et al., Nat Protoc 2014;9:209-22, the entire contents of which are incorporated herein by reference). As expected, TSLP / OVA administration induced an increase in lung tissue eosinophils at comparable levels across mouse strains (Figure 4A). Furthermore, TSLP / OVA induced comparable levels of pulmonary expression of Muc5ac (Figure 4B) and comparable levels of circulating levels of Ova-specific IgE (Figure 4C) and Ova-specific IgG1 (Figure 4D).

[0212] method Ova-alum-induced pulmonary inflammation. 50 μg OVA (grade V, Sigma Aldrich) emulsified with 2 mg aluminum hydroxide (Sigma Aldrich) or 2 mg aluminum hydroxide alone were administered intraperitoneally to WT Balb / c mice on days 1 and 14. Anesthetized mice were challenged intranasally with 150 μg OVA (grade III, Sigma Aldrich) in 20 μL PBS for 4 days, starting on day 21. Mice were analyzed 24 h after the final challenge.

[0213] At the end of the study, mice were sacrificed and blood and lungs were collected for analysis of eosinophil infiltration in lung tissue, pulmonary gene expression by real-time qPCR, and circulating serum immunoglobulin levels.

[0214] Flow cytometry analysis. To allow flow cytometry analysis of circulating and tissue-infiltrating immune cells in the lungs, mice were intravenously injected with anti-CD45 BV650 antibody (BD Biosciences) 5 min before sacrifice to selectively label immune cells still in the vasculature, while leaving cells that had infiltrated the lung parenchyma unlabeled. Mouse caudal lung lobes were digested and single-cell suspensions were prepared using a solution of Liberase TH (Roche) and DNase I (Roche), followed by mechanical dissociation. Cells were then stained with LIVE / DEAD Fixable Dead Cell Stain (BD Biosciences) to remove dead cells and then treated with antibodies against: CD45, CD26, Siglec-F, Ly6G, Ly6C, CD11b, CD19, SIRPα, CD23, CD127, Sca-1, CD44, CD4, CD8, TCRb, CD69 CD62L (BD Biosciences); CD64, XCR1, IA / IE, CD11c, CD301b (Biolegend); MerTK, ST2 (eBioscience). Samples were acquired on an LSR FortessaX-20 or FACSymphony cell analyzer using an HTS attachment (BD). Data analysis was performed using FlowJov10 Software (BD).

[0215] Measurement of serum IgE and antigen-specific IgE or IgG1. Whole blood was collected into Microtainer SST serum tubes and pelleted by centrifugation at 15,000 g for 10 min at 4° C. Serum samples were used to determine concentrations: total IgE concentration by IgE sandwich ELISA OptEIA kit (BD Biosciences), total anti-Ova IgE and anti-Ova IgG1 concentrations by indirect ELISA (Chondrex), total anti-HDM IgE by sandwich ELISA (Chondrex), and total anti-HDM IgG1 titers by in-house sandwich ELISA. For all ELISA kits, manufacturer's instructions were followed.

[0216] Measurement of Muc5ac. Lung Muc5ac gene expression was detected in harvested lung tissue by real-time qPCR and normalized to housekeeping genes. Briefly, at the end of the study, mice were exsanguinated and the right lung accessory lobe was removed from each mouse and then placed in a tube containing 400 μL of RNA and stored at -20°C. All samples were homogenized in TRIzol and chloroform was used for phase separation. The aqueous phase containing total RNA was purified using MagMAX™-96 for Microarrays Total RNA Isolation Kit (Ambion by Life Technologies) according to the manufacturer's specifications. Genomic DNA was removed using RNase-Free DNase Set (Qiagen). mRNA was reverse transcribed to cDNA using SuperScript® VILO™ Master Mix (Invitrogen by Life Technologies). cDNA was diluted to 2ng / uL and 10ng cDNA was amplified with SensiFAST Probe Hi-ROX (Meridian) using an ABI 7900HT Sequence Detection System (Applied Biosystems). An internal control housekeeping gene was used to normalize for differences in cDNA input. Fold changes in lung tissue mRNA expression levels relative to control mice were measured and expressed relative to housekeeping mRNA expression. [Table 13]

[0217] conclusion The Ova-Alum model was developed based on the analysis of pulmonary eosinophil infiltration, pulmonary gene expression analysis, and circulating antibody levels in a double humanized mouse model (Tslp hu / hu / Tslpr hu / hu ) and triple humanized mice (Tslp hu / hu / Tslpr hu / hu / Il7ra hu / hu ) induced similar levels of type 2-driven inflammation.

Claims

1. A genetically modified rodent, or isolated rodent tissue or cell, comprising in its genome: a rodent Tslpr nucleic acid sequence encoding a transmembrane cytoplasmic sequence having at least 95% identity to the transmembrane cytoplasmic sequence of an endogenous rodent Tslpr protein; a human TSLPR nucleic acid sequence encoding at least a substantial portion of the extracellular domain of a human TSLPR protein, optionally said human TSLPR nucleic acid sequence comprising exon 2 up to the codon for the last extracellular domain amino acid in exon 6 of the human TSLPR gene; the humanized Tslpr gene encodes a humanized Tslpr polypeptide comprising an extracellular domain having at least 95% identity to the extracellular domain of the human TSLPR protein, and a transmembrane cytoplasmic sequence having at least 95% identity to the transmembrane cytoplasmic sequence of the endogenous rodent Tslpr protein, and optionally a signal peptide of the endogenous rodent Tslpr protein; the humanized Tslpr gene is operably linked to an endogenous rodent Tslpr promoter and expresses the humanized Tslpr polypeptide in the genetically modified rodent; the rodent is a mouse or a rat; Optionally, the genetically modified rodent, or the isolated rodent tissue or cell, is homozygous for the humanized Tslpr gene; Optionally, the genetically modified rodent is a mouse, and the humanized Tslpr gene comprises (i) exon 1 of the mouse Tslpr gene, (ii) exon 2 to the codon of the last amino acid of the extracellular domain in exon 6 of the human TSLPR gene, and (iii) exon 6 from the codon of the first amino acid of the transmembrane domain to exon 8 of the mouse Tslpr gene. Genetically modified rodents, or isolated rodent tissues or cells.

2. the humanized Tslpr gene is located at the endogenous rodent Tslpr locus; Optionally, the humanized Tslpr gene is formed as a result of replacement of rodent genomic DNA comprising an exon sequence encoding at least a substantial portion of the extracellular domain of the endogenous rodent Tslpr protein with the human TSLPR nucleic acid encoding at least a substantial portion of the extracellular domain of the human TSLPR protein.

2. A genetically modified rodent, or isolated rodent tissue or cell, as described in claim 1.

3. 3. The genetically modified rodent, or isolated rodent tissue or cell of claim 1 or 2, whose genome further comprises a humanized Tslp gene at the endogenous rodent Tslp locus, a humanized Il7ra gene at the endogenous rodent Il7ra locus, or a combination thereof.

4. The isolated rodent tissue or cell of any one of claims 1 to 3, wherein the rodent cell is a rodent embryonic stem cell.

5. A rodent embryo comprising the rodent embryonic stem cell of claim 4.

6. 1. A method for producing a genetically modified rodent, comprising: introducing into the genome of a rodent embryonic stem (ES) cell a nucleic acid molecule comprising a human TSLPR nucleic acid sequence, said human TSLPR nucleic acid sequence encoding at least a substantial portion of an extracellular domain of a human TSLPR protein, optionally said human TSLPR nucleic acid sequence comprising exon 2 up to the codon for the last extracellular domain amino acid in exon 6 of the human TSLPR gene; obtaining a rodent ES cell in which the human TSLPR nucleic acid sequence has been integrated into the endogenous Tslpr locus to replace rodent Tslpr genomic DNA, thereby forming the humanized Tslpr gene, wherein the humanized Tslpr gene encodes a humanized Tslpr polypeptide comprising an extracellular domain that is at least 95% identical to the extracellular domain of the human TSLPR protein, and a transmembrane cytoplasmic sequence that has at least 95% identity to the transmembrane cytoplasmic sequence of the endogenous rodent Tslpr protein, and optionally a signal peptide of the endogenous rodent Tslpr protein, wherein the humanized Tslpr gene is operably linked to an endogenous rodent Tslpr promoter; generating said genetically modified rodent from said obtained rodent ES cells; the rodent is a mouse or a rat; Optionally, the genetically modified rodent is a mouse, and the humanized Tslpr gene comprises (i) exon 1 of the endogenous mouse Tslpr gene, (ii) exon 2 to the codon for the last amino acid of the extracellular domain in exon 6 of the human TSLPR gene, and (iii) exon 6 from the codon for the first amino acid of the transmembrane domain to exon 8 of the endogenous mouse Tslpr gene. method.

7. A genetically modified rodent, or isolated rodent tissue or cell, comprising in its genome: a rodent Tslp nucleic acid sequence comprising an exon sequence of an endogenous rodent Tslp gene encoding an endogenous rodent Tslp signal peptide; a human TSLP nucleic acid sequence from a codon in exon 1 of the human TSLP gene for the first amino acid of the mature protein sequence of the human TSLP protein to a stop codon in exon 4 of said human TSLP gene, the humanized Tslp gene encodes a humanized Tslp polypeptide comprising an endogenous rodent Tslp signal peptide and the mature protein sequence of the human TSLP protein; the humanized Tslp gene is operably linked to an endogenous rodent Tslp promoter, and expresses the humanized Tslp polypeptide in the genetically modified rodent; the rodent is a mouse or a rat; Optionally, the humanized Tslp gene comprises the 3'UTR of the endogenous rodent Tslp gene; Optionally, the genetically modified rodent, or the isolated rodent tissue or cell, is homozygous for the humanized Tslp gene; Optionally, the genetically modified rodent is a mouse, and the humanized Tslp gene comprises (i) exon 1, encoding a signal peptide amino acid, and a 5' portion of exon 2, of the mouse Tslp gene, (ii) exon 1 from the codon for the first amino acid of the mature protein sequence of the human TSLP gene to a stop codon in exon 4, and (iii) the 3'UTR of the mouse Tslp gene. Genetically modified rodents, or isolated rodent tissues or cells.

8. The genetically modified rodent or isolated rodent tissue or cell of claim 7, wherein the humanized Tslp gene is located at an endogenous rodent Tslp locus, and optionally the humanized Tslp gene is formed as a result of replacement of rodent genomic DNA comprising an exon sequence encoding a mature protein sequence of the endogenous rodent Tslp protein with the human TSLP nucleic acid encoding the mature protein sequence of the human TSLP protein.

9. A genetically modified rodent, or isolated rodent tissue or cell, as described in claim 7 or 8, whose genome further comprises a humanized Tslpr gene at the endogenous rodent Tslpr locus, a humanized Il7ra gene at the endogenous rodent Il7ra locus, or a combination thereof.

10. The isolated rodent cell of any one of claims 7 to 9, wherein the rodent cell is a rodent embryonic stem cell.

11. A rodent embryo comprising the rodent embryonic stem cell described in claim 10.

12. A method for producing a genetically modified rodent, comprising: introducing into the genome of a rodent embryonic stem (ES) cell a nucleic acid molecule comprising a human TSLP nucleic acid sequence, said human TSLP nucleic acid sequence comprising a codon in exon 1 of the human TSLP gene for the first amino acid of a mature protein sequence of a human TSLP protein through a stop codon in exon 4 of said human TSLP gene; obtaining a rodent ES cell in which the human TSLP nucleic acid sequence has been integrated into an endogenous rodent Tslp locus to replace rodent Tslp genomic DNA, thereby forming a humanized Tslp gene, the humanized Tslp gene encoding a humanized Tslp polypeptide comprising an endogenous rodent Tslp signal peptide and the mature protein sequence of the human TSLP protein, the humanized Tslp gene being operably linked to an endogenous rodent Tslp promoter; generating said genetically modified rodent from said obtained rodent ES cells; The rodent is a mouse or a rat; method.

13. A genetically modified rodent, or isolated rodent tissue or cell, comprising in its genome: comprising a humanized Il7ra gene, the humanized Il7ra gene encodes a humanized Il7ra polypeptide comprising: (i) a signal peptide of an endogenous rodent Il7ra protein; (ii) a substantial portion of an extracellular domain of a human IL7RA protein, optionally wherein said substantial portion is defined by amino acids 21-236 of SEQ ID NO:43; (iii) the last two amino acids of the extracellular domain of said endogenous Il7ra protein; and (iv) a transmembrane cytoplasmic sequence of said endogenous rodent Il7ra protein; the humanized Il7ra gene is operably linked to an endogenous rodent Il7ra promoter and expresses the humanized Il7ra polypeptide in the genetically modified rodent; the rodent is a mouse or a rat; Optionally, the humanized IL7ra gene comprises the 5'UTR of exon 1 of the endogenous rodent IL7ra gene; Optionally, the genetically modified rodent, or the isolated rodent tissue or cell, is homozygous for the humanized Il7ra gene; Optionally, the genetically modified rodent is a mouse, and the humanized Il7ra gene comprises (i) a portion of exon 1 of the mouse Il7ra gene, including a 5'UTR and a sequence encoding the signal peptide of the mouse Il7ra, (ii) from the codon of exon 1 of the human IL7RA gene encoding the first amino acid of a mature human IL7RA protein through exon 5 of the human IL7RA gene, and (iii) exon 6 through exon 8 of the mouse Il7ra gene. Genetically modified rodents, or isolated rodent tissues or cells.

14. the humanized Il7ra gene is located at the endogenous rodent Il7ra locus; Optionally, the humanized Il7ra gene is formed as a result of replacement of rodent Il7ra genomic DNA at an endogenous rodent Il7ra locus with a human IL7RA nucleic acid; Optionally, the genetically modified rodent is a mouse, and the mouse genomic DNA to be replaced comprises a codon in exon 1 through exon 5 of the endogenous mouse Il7ra gene that encodes a first amino acid of a mature mouse Il7ra protein, and the human genomic DNA comprises a codon in exon 1 through exon 5 of the human IL7RA gene that encodes the first amino acid of the mature human IL7RA protein.

14. A genetically modified rodent, or isolated rodent tissue or cell, as described in claim 13.

15. 15. The genetically modified rodent, or isolated rodent tissue or cell of claim 13 or 14, whose genome further comprises a humanized Tslp gene at the endogenous rodent Tslp locus, a humanized Tslpr gene at the endogenous rodent Tslpr locus, or a combination thereof.

16. The isolated rodent cell of any one of claims 13 to 15, wherein the rodent cell is a rodent embryonic stem cell.

17. A rodent embryo comprising the rodent embryonic stem cell of claim 16.

18. 1. A method for producing a genetically modified rodent, comprising: introducing into the genome of a rodent embryonic stem (ES) cell a nucleic acid molecule comprising a human IL7RA nucleic acid sequence, said human IL7RA nucleic acid sequence encoding at least a substantial portion of the extracellular domain of said human IL7RA protein; obtaining a rodent ES cell in which the human IL7RA nucleic acid sequence has been integrated into an endogenous Il7ra locus to replace rodent Il7ra genomic DNA, thereby forming the humanized Il7ra gene, wherein the humanized Il7ra gene encodes a humanized Il7ra polypeptide comprising: (i) a signal peptide of an endogenous rodent Il7ra protein; (ii) a substantial portion of the extracellular domain of a human IL7RA protein, optionally wherein the substantial portion is defined by amino acids 21-236 of SEQ ID NO:43; (iii) the last two amino acids of the extracellular domain of the endogenous rodent Il7ra protein; and (iv) a transmembrane cytoplasmic sequence of the endogenous Il7ra protein, wherein the humanized Tslpr gene is operably linked to an endogenous rodent Il7ra promoter; generating said genetically modified rodent from said obtained rodent ES cells; The rodent is a mouse or a rat; method.

19. 16. The genetically modified rodent of any one of claims 1-3, 7-9 or 13-15, further comprising a humanized Sirpα gene, wherein the genetically modified rodent is homozygous for RAG2- / - and IL2RG- / -, and optionally, wherein the genetically modified rodent further comprises a humanized Tpo gene, and / or a humanized GM-CSF / IL-3 locus.

20. 1. A method for testing a candidate agent for treating an allergic condition, comprising: Inducing an allergic condition in a genetically modified rodent as defined by any of claims 1 to 3, 7 to 9, 13 to 15, or 19; administering a candidate agent to the genetically modified rodent, optionally wherein the candidate agent is a small molecule compound, a nucleic acid, or an antibody; and determining whether the candidate agent inhibits the allergic condition in the genetically modified rodent.

21. 1. A method for testing a candidate drug for treating cancer, comprising: Transplanting human cancer cells into a genetically modified rodent as defined by any one of claims 1 to 3, 7 to 9, 13 to 15, or 19; administering a candidate agent to the genetically modified rodent, optionally wherein the candidate agent is a small molecule compound, a nucleic acid, or an antibody; and determining whether the candidate agent inhibits proliferation of the cancer cells in the genetically modified rodent.

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