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CN-122012617-A - MET gene modified non-human animal

CN122012617ACN 122012617 ACN122012617 ACN 122012617ACN-122012617-A

Abstract

The invention belongs to the field of transgenic animals, and relates to a non-human animal modified by MET gene. In particular, the present invention provides a method of making a genetically modified non-human animal comprising modifying a non-human animal genome such that the modified non-human animal genome comprises a nucleotide sequence encoding a human or chimeric hepatocyte growth factor receptor (MET) protein. Also provided is a genetically modified non-human animal genome comprising a nucleotide sequence encoding a human or chimeric MET protein. The non-human animal obtained by the method provides a powerful tool for researching the function of MET protein and provides a platform for screening related medicines.

Inventors

  • LI CHONG
  • ZHANG ZHI
  • GUO JING

Assignees

  • 百奥赛图(北京)医药科技股份有限公司

Dates

Publication Date
20260512
Application Date
20260228
Priority Date
20250228

Claims (14)

  1. 1. A method of making a genetically modified non-human animal, the method comprising modifying a non-human animal genome such that the modified non-human animal genome comprises a nucleotide sequence encoding a human or chimeric hepatocyte growth factor receptor (MET) protein; Preferably, the chimeric MET protein comprises (i) an extracellular or chimeric extracellular region of a human MET protein (human or chimeric extracellular region), (ii) a transmembrane region (endogenous transmembrane region) of an endogenous non-human animal MET protein, and (iii) a cytoplasmic region (endogenous cytoplasmic region) of an endogenous non-human animal MET protein; Preferably, the chimeric MET protein further comprises a signal peptide (endogenous signal peptide) of an endogenous non-human animal MET protein; Preferably, the nucleotide sequence encoding a human or chimeric MET protein is located at an endogenous non-human animal MET locus; preferably, wherein the modification comprises inserting a nucleotide sequence of a human MET gene into the endogenous non-human animal MET locus of the non-human animal to form the nucleotide sequence encoding a human or chimeric MET protein at the endogenous non-human animal MET locus.
  2. 2. The method of claim 1, wherein the step of, The nucleotide sequence of the human MET gene replaces the nucleotide sequence at the endogenous non-human animal MET locus; Preferably, the nucleotide sequence of the human MET gene is selected from the group consisting of nucleotide sequences encoding all or part of the extracellular region of a human MET protein; preferably, the nucleotide sequence of the human MET gene comprises at least 1000-10000, 10000-50000, 50000-100000bp nucleotides of the part of exon 2, all of exon 3 to exon 12 and the part of exon 13 of the human MET gene, or the part of exon 2, all of exon 3 to exon 12 and the part of exon 13 of the human MET gene; Preferably, the nucleotide sequence at the endogenous non-human animal MET locus that is replaced comprises a nucleotide sequence encoding all or part of the extracellular region of an endogenous MET protein; Preferably, the nucleotide sequence at the endogenous non-human animal MET locus that is replaced comprises a portion of exon 3, all of exons 4 to 13, and a portion of exon 14 of the endogenous non-human animal (e.g., mouse) MET gene; Preferably, after the substitution, the genomic sequence of the remaining endogenous non-human animal (e.g., mouse) MET gene comprises all of exons 1 to 2, part of exon 3, part of exon 14, and all of exons 15 to 22 of the endogenous non-human animal MET gene; Or alternatively The nucleotide sequence encoding the chimeric MET protein comprises all of non-human animal MET gene exons 1 to 2, a portion of non-human animal MET gene exons 3, a portion of human MET gene exons 2, all of human MET gene exons 3 to 12, a portion of human MET gene exons 13, a portion of non-human animal MET gene exons 14, and all of non-human animal MET gene exons 15 to 22; Or alternatively The amino acid sequence of the human or chimeric MET protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity as compared to amino acids at positions 25-932 of SEQ ID NO. 2, or 31-915 of SEQ ID NO. 2; Preferably, the amino acid sequence of the chimeric MET protein comprises the amino acid sequence set forth in SEQ ID 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence set forth in SEQ ID 10; Or alternatively The nucleotide sequence encoding a human or chimeric MET protein comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the nucleotide sequence at position 116699175-116771512 of NCBI accession nc_ 000007.14; preferably, the nucleotide sequence encoding a chimeric MET protein comprises the nucleotide sequence set forth in SEQ ID NO. 5, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO. 5; Or alternatively The nucleotide sequence encoding a human or chimeric MET protein is operably linked to an endogenous MET regulatory element (e.g., an endogenous promoter, 5'utr, and/or 3' utr); Or alternatively The genetically modified non-human animal does not express endogenous MET protein or has reduced expression levels compared to MET in a wild-type animal; Or alternatively The genetically modified non-human animal genome further comprises a nucleotide sequence encoding a human or chimeric HGF protein; Preferably, the amino acid sequence of the human or chimeric HGF protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO:21, SEQ ID NO:21 positions 32-728, SEQ ID NO:21 positions 33-728, SEQ ID NO:20 positions 1-31, SEQ ID NO:20 positions 1-32; Preferably, the amino acid sequence of the human or chimeric HGF protein comprises the amino acid sequence shown as SEQ ID NO. 30, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the amino acid sequence shown as SEQ ID NO. 30; Preferably, the nucleotide sequence encoding a human or chimeric HGF protein comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the nucleotide sequence shown in SEQ ID NO. 24; preferably, the nucleotide sequence encoding a human or chimeric HGF protein comprises a nucleotide sequence as set forth in SEQ ID NO. 25; Or alternatively The method further comprises the steps of: Inserting a nucleotide sequence of a human HGF gene into the endogenous non-human animal HGF locus of the non-human animal to form the nucleotide sequence encoding a human or chimeric MET protein at the endogenous non-human animal HGF locus; Or alternatively The non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by an additional gene selected from at least one of HGF, EGFR, FCRN, LAG, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA 4; Or alternatively The non-human animal is selected from a mammal, such as a monkey, a rodent, preferably the rodent is selected from a mouse or a rat, preferably a mouse.
  3. 3. A genetically modified non-human animal genome comprising a nucleotide sequence encoding a human or chimeric hepatocyte growth factor receptor (MET) protein.
  4. 4. The genetically modified non-human animal genome of claim 3, wherein, The chimeric MET proteins comprise (i) an extracellular or chimeric extracellular region of a human MET protein (human or chimeric extracellular region), (ii) a transmembrane region (endogenous transmembrane region) of an endogenous non-human animal MET protein, and (iii) a cytoplasmic region (endogenous cytoplasmic region) of an endogenous non-human animal MET protein; Preferably, the chimeric MET protein further comprises a signal peptide (endogenous signal peptide) of an endogenous non-human animal MET protein; Or alternatively The human or chimeric extracellular region comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95% or 100% identity to the amino acid sequence at positions 25-932 of SEQ ID NO. 2; Preferably, the human or chimeric extracellular region comprises an amino acid sequence having at least 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 850, 880, 884, 885, 890, 905 or 908 consecutive amino acid identity with the amino acid sequence at positions 25-932 of SEQ ID NO. 2; preferably, the human or chimeric extracellular region comprises the amino acid sequence at positions 31-915 of SEQ ID NO. 2; Preferably, the chimeric extracellular region comprises the amino acid sequence at positions 25-30 of SEQ ID NO. 1, and the amino acid sequence at positions 31-915 of SEQ ID NO. 2, the amino acid sequence at positions 915-931 of SEQ ID NO. 1; Preferably, the chimeric extracellular region comprises the amino acid sequence at positions 25-932 of SEQ ID NO. 10; Or alternatively Preferably, the rodent is selected from a mouse or a rat, preferably a mouse; Or alternatively Said non-human animal is selected from the group consisting of mice, and wherein optionally said endogenous transmembrane region comprises amino acid sequences 932-954 of SEQ ID No. 1; Or alternatively The non-human animal is selected from the group consisting of mice, and wherein optionally the endogenous cytoplasmic region comprises the amino acid sequences 955-1379 of SEQ ID NO. 1; Or alternatively The non-human animal is selected from a mouse, and wherein optionally the endogenous signal peptide comprises the amino acid sequence at positions 1-24 of SEQ ID No. 1; Or alternatively The human or chimeric MET protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 2; Or alternatively The nucleotide sequence encoding a human or chimeric MET protein is located at an endogenous non-human animal MET locus; Or alternatively The nucleotide sequence at the endogenous non-human animal MET locus is replaced with the nucleotide sequence of a human MET gene; Further preferably, the nucleotide sequence of the human MET gene is selected from the group consisting of nucleotide sequences encoding all or part of the extracellular region of human MET protein; Further preferably, the nucleotide sequence of the human MET gene comprises at least 1000-10000, 10000-50000, 50000-100000bp nucleotides of the part of exon 2, all of exon 3 to exon 12 and the part of exon 13 of the human MET gene, or the part of exon 2, all of exon 3 to exon 12 and the part of exon 13 of the human MET gene; Or alternatively The non-human animal is a mouse comprising any one selected from the group consisting of: (1) At the endogenous mouse MET locus, a nucleotide sequence encoding a human MET protein, or (2) At an endogenous mouse MET locus, a nucleotide sequence encoding a chimeric MET protein, wherein the nucleotide sequence encoding a chimeric MET protein comprises, in 5 '-3', all of non-human animal MET gene exons 1 to 2, a portion of non-human animal MET gene exons 3, a portion of human MET gene exons 2, all of human MET gene exons 3 to 12, a portion of human MET gene exons 13, a portion of non-human animal MET gene exons 14, and all of non-human animal MET gene exons 15 to 22; Preferably, the amino acid sequence of the chimeric MET protein comprises the amino acid sequence set forth in SEQ ID 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence set forth in SEQ ID 10; Preferably, the nucleotide sequence encoding a chimeric MET protein comprises the nucleotide sequence set forth in SEQ ID NO. 5, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO. 5; Or alternatively The nucleotide sequence encoding a human or chimeric MET protein is operably linked to an endogenous regulatory element (e.g., an endogenous promoter, 5'utr, and/or 3' utr) of an endogenous non-human animal MET locus; Or alternatively The non-human animal does not express an endogenous MET protein or has a reduced expression level of an endogenous MET protein compared to a wild-type non-human animal; Or alternatively The modified MET gene in the non-human animal genome is homozygous or heterozygous for the endogenous replaced locus; Or alternatively The genetically modified non-human animal genome further comprises a nucleotide sequence encoding a human or chimeric HGF protein; Preferably, the amino acid sequence of the human or chimeric HGF protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO:21, SEQ ID NO:21 positions 32-728, SEQ ID NO:21 positions 33-728, SEQ ID NO:20 positions 1-31, SEQ ID NO:20 positions 1-32; Preferably, the amino acid sequence of the human or chimeric HGF protein comprises the amino acid sequence shown as SEQ ID NO. 30, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the amino acid sequence shown as SEQ ID NO. 30; Preferably, the nucleotide sequence encoding a human or chimeric HGF protein comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the nucleotide sequence shown in SEQ ID NO. 24; preferably, the nucleotide sequence encoding a human or chimeric HGF protein comprises a nucleotide sequence as set forth in SEQ ID NO. 25; Or alternatively The genetically modified non-human animal genome further comprises a nucleotide sequence of a human or chimeric protein encoded by an additional gene selected from at least one of EGFR, FCRN, LAG, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA 4.
  5. 5. A method of constructing a genetically modified non-human animal cell, the method comprising replacing, at an endogenous non-human animal (e.g., mouse) MET locus, a nucleotide sequence encoding an endogenous MET protein with a nucleotide sequence encoding a human or chimeric MET protein, producing a genetically modified non-human animal cell that expresses a human or chimeric MET protein.
  6. 6. The method of claim 5, wherein, The chimeric MET protein comprises a human or chimeric extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region; preferably, the human or chimeric extracellular region comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95% or 100% identity to the extracellular region of a human MET protein; Or alternatively The nucleotide sequence encoding a human or chimeric MET protein comprises a portion of exon 2, all of exons 3 to 12, and a portion of exon 13 of a human MET gene; Preferably, the nucleotide sequence encoding a human or chimeric MET protein comprises at least 1000-10000, 10000-50000, 50000-100000 nucleotides of the portion of exon 2, all of exon 3 to exon 12, and the portion of exon 13 of the human MET gene; Or alternatively The human or chimeric MET protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 2, SEQ ID NO. 31-915 or SEQ ID NO. 2 at positions 25-932; Or alternatively The nucleotide sequence encoding a human or chimeric MET protein comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the nucleotide sequence at position 116699175-116771512 of NCBI accession nc_ 000007.14; Or alternatively The nucleotide sequence encoding an endogenous MET protein comprises a nucleotide sequence encoding all or a portion of an extracellular region of an endogenous MET protein; Or alternatively The nucleotide sequence encoding an endogenous MET protein comprises a portion of exon 3, all of exons 4 to 13, and a portion of exon 14 of a non-human animal (e.g., mouse) MET gene; Or alternatively The nucleotide sequence encoding the chimeric MET protein comprises all of non-human animal MET gene exons 1 to 2, a portion of non-human animal MET gene exons 3, a portion of human MET gene exons 2, all of human MET gene exons 3 to 12, a portion of human MET gene exons 13, a portion of non-human animal MET gene exons 14, and all of non-human animal MET gene exons 15 to 22; Preferably, the amino acid sequence of the chimeric MET protein comprises the amino acid sequence set forth in SEQ ID 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence set forth in SEQ ID 10; preferably, the nucleotide sequence encoding a human or chimeric MET protein is operably linked to regulatory elements of an endogenous MET (e.g., an endogenous promoter, 5'utr, and/or 3' utr); Or alternatively The non-human animal is a mouse; Or alternatively The non-human animal further comprises a nucleotide sequence encoding a human or chimeric HGF protein; Preferably, the amino acid sequence of the human or chimeric HGF protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO:21, SEQ ID NO:21 positions 32-728, SEQ ID NO:21 positions 33-728, SEQ ID NO:20 positions 1-31, SEQ ID NO:20 positions 1-32 or SEQ ID NO: 30; Preferably, the nucleotide sequence encoding a human or chimeric HGF protein comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the nucleotide sequence set forth in SEQ ID NO. 24; preferably, the nucleotide sequence encoding a human or chimeric HGF protein comprises a nucleotide sequence as set forth in SEQ ID NO. 25; Or alternatively The non-human animal also includes nucleotide sequences of human or chimeric proteins encoded by other genes selected from at least one of HGF, EGFR, FCRN, LAG, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA 4.
  7. 7. A method of determining the effectiveness of a therapeutic agent in treating cancer, the method comprising: 1) Providing a non-human animal obtainable by the method of claim 1 or 2, or a non-human animal comprising the genetically modified non-human animal genome of claim 3 or 4; 2) Administering a therapeutic agent to said animal, wherein said animal has cancer; 3) Determining the inhibition of cancer by the therapeutic agent; preferably, the therapeutic agent is an anti-human MET and/or HGF antibody, an oligonucleotide drug, and/or a polypeptide drug; Preferably, the cancer is a tumor and the inhibition of the tumor by the therapeutic agent is determined by measuring the tumor volume of the animal; Preferably, the animal is afflicted with cancer by injecting one or more cancer cells into the animal; preferably, the cancer is a solid tumor or hematological tumor, preferably, the cancer is non-small cell lung cancer, colorectal cancer, gastric cancer, head and neck cancer, liver cancer, or lung cancer.
  8. 8. A method of determining the effectiveness of a therapeutic agent and an additional therapeutic agent in treating cancer, the method comprising: 1) Providing a non-human animal obtainable by the method of claim 1 or 2, or a non-human animal comprising the genetically modified non-human animal genome of claim 3 or 4; 2) Administering a therapeutic agent and an additional therapeutic agent to the animal, wherein the animal has a tumor; 3) Determining the inhibition of the tumor by the therapeutic agent and the additional therapeutic agent; Preferably, the therapeutic agent is an anti-human MET and/or HGF antibody, an oligonucleotide drug, and/or a polypeptide drug; Preferably, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA 4 antibody; preferably, the animal has a tumor by injecting one or more cancer cells into the animal; Preferably, the cancer is a tumor and the inhibition of the tumor by the therapeutic agent and the additional therapeutic agent is determined by measuring the tumor volume of the animal; preferably, the cancer is a solid tumor or hematological tumor, preferably, the cancer is non-small cell lung cancer, colorectal cancer, gastric cancer, head and neck cancer, liver cancer, or lung cancer.
  9. 9. A method of determining the effectiveness of a therapeutic agent in treating an immune disorder, the method comprising: 1) Providing a non-human animal obtainable by the method of claim 1 or 2, or a non-human animal comprising the genetically modified non-human animal genome of claim 3 or 4; 2) Administering a therapeutic agent to the animal, wherein the non-human animal suffers from an immune disease; 3) The therapeutic effect of the therapeutic agent on an immune disorder is determined, preferably, the immune disorder is asthma, rheumatoid arthritis or multiple sclerosis.
  10. 10. A method of determining the effectiveness of a therapeutic agent in treating inflammation, the method comprising: 1) Providing a non-human animal obtainable by the method of claim 1 or 2, or a non-human animal comprising the genetically modified non-human animal genome of claim 3 or 4; 2) Administering a therapeutic agent to said animal, wherein said animal has inflammation; 3) Determining the effectiveness of the therapeutic agent for treating the inflammation; preferably, the inflammation is Inflammatory Bowel Disease (IBD).
  11. 11. A method of determining toxicity of a therapeutic agent, the method comprising: 1) Providing a non-human animal obtainable by the method of claim 1 or 2, or a non-human animal comprising the genetically modified non-human animal genome of claim 3 or 4; 2) Administering a therapeutic agent to said animal; 3) Determining the effect of the therapeutic agent on the animal; Preferably, the determining the effect of the therapeutic agent on the animal includes, but is not limited to, measuring the weight, erythrocyte count, hematocrit, and/or hemoglobin of the animal.
  12. 12. A humanized MET protein, comprising all or a portion of the amino acid sequence of a human MET protein; Preferably, the humanized MET protein comprises (i) an extracellular or chimeric extracellular region of a human MET protein (human or chimeric extracellular region), (ii) a transmembrane region of an endogenous non-human animal MET protein (endogenous transmembrane region), and (iii) a cytoplasmic region of an endogenous non-human animal MET protein (endogenous cytoplasmic region); Preferably, the chimeric MET protein further comprises a signal peptide (endogenous signal peptide) of an endogenous non-human animal MET protein; Preferably, the human or chimeric extracellular region comprises the amino acid sequence at positions 31-915 of SEQ ID NO. 2, preferably, the chimeric extracellular region comprises the amino acid sequence at positions 25-30 of SEQ ID NO. 1, and the amino acid sequence at positions 31-915 of SEQ ID NO. 2, the amino acid sequence at positions 915-931 of SEQ ID NO. 1; Preferably, the non-human animal is selected from a mammal, such as a monkey, a rodent, preferably the rodent is selected from a mouse or a rat, preferably a mouse, preferably the humanized MET protein comprises an amino acid sequence set forth in SEQ ID NO. 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 10.
  13. 13. An isolated nucleic acid molecule or a humanized MET gene comprising a nucleotide sequence encoding the humanized MET protein of claim 56 or 57; Preferably, the isolated nucleic acid molecule or humanized MET gene comprises a portion of exon 2, all of exon 3 to exon 12 and a portion of exon 13 of a human MET gene, said isolated nucleic acid molecule or humanized MET gene further comprising all of exon 1 to exon 2, all of exon 3, part of exon 14 and all of exon 15 to exon 22 of a non-human animal MET gene; Preferably, the isolated nucleic acid molecule or humanized MET gene comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the nucleotide sequence set forth in SEQ ID NO 3, 4,5, 6, 7, 8 or 9; Preferably, the isolated nucleic acid molecule or humanized MET gene comprises the nucleotide sequence set forth in SEQ ID NO. 5.
  14. 14. A cell, tissue or organ comprising the humanized MET protein of claim 12, the isolated nucleic acid molecule or humanized MET gene of claim 13, or the humanized MET genome of claim 3 or 4.

Description

MET gene modified non-human animal Technical Field The present invention provides a non-human animal expressing human or chimeric (e.g., humanized) MET proteins and methods of use thereof. Background In vitro screening methods are generally used in traditional drug development, however, these screening methods cannot provide body environment (such as tumor microenvironment, stromal cells, extracellular matrix components, immune cell interactions, etc.), resulting in higher failure rate in drug development. Furthermore, given the differences between humans and animals, the experimental results obtained from in vivo pharmacological tests using conventional laboratory animals may not reflect the actual disease state and interaction at the targeted site, resulting in significant differences between the results of many clinical trials and animal experimental results. Therefore, developing humanized animal models suitable for human antibody screening and evaluation will significantly improve the development efficiency of new drugs and reduce the cost of drug development. Disclosure of Invention SUMMARY The present application provides an animal model with human or chimeric hepatocyte growth factor receptor (MET) and/or Hepatocyte Growth Factor (HGF) proteins and a method for preparing the same. The animal model can express human or chimeric MET (e.g., humanized MET), and/or human or chimeric HGF (e.g., humanized HGF) protein. It is useful for the study of MET and/or HGF gene function, and also for the screening and evaluation of MET/HGF signaling pathway modulators (e.g., anti-human MET and/or HGF antibodies, oligonucleotide drugs, and/or polypeptide drugs). In addition, the animal model prepared by the method can be used for drug screening, pharmacodynamics research, treatment of immune related diseases and treatment of diseases of human MET/HGF target sites, and can also be used for promoting development and design of new drugs, thereby saving time and cost. In conclusion, the application provides a powerful tool for researching the function of MET protein and provides a platform for screening related medicines. In one aspect, the invention provides a method of making a genetically modified non-human animal comprising modifying a non-human animal genome such that the modified non-human animal genome comprises a nucleotide sequence encoding a human or chimeric hepatocyte growth factor receptor (MET) protein. In some embodiments, the chimeric MET protein comprises (i) an extracellular or chimeric extracellular region of a human MET protein (human or chimeric extracellular region), (ii) a transmembrane region (endogenous transmembrane region) of an endogenous non-human animal MET protein, and (iii) a cytoplasmic region (endogenous cytoplasmic region) of an endogenous non-human animal MET protein. In some embodiments, the chimeric MET protein further comprises a signal peptide (endogenous signal peptide) of an endogenous non-human animal MET protein. In some embodiments, the nucleotide sequence encoding a human or chimeric MET protein is located at an endogenous non-human animal MET locus. In some embodiments, wherein the modification comprises inserting a nucleotide sequence of a human MET gene into the endogenous non-human animal MET locus of the non-human animal to form the nucleotide sequence encoding a human or chimeric MET protein at the endogenous non-human animal MET locus. In some embodiments, the nucleotide sequence of the human MET gene replaces the nucleotide sequence at the endogenous non-human animal MET locus. In some embodiments, the nucleotide sequence of the human MET gene is selected from the group consisting of nucleotide sequences encoding all or a portion of the extracellular region of a human MET protein. In some embodiments, the nucleotide sequence of the human MET gene comprises at least 1000-10000, 10000-50000, 50000-100000bp nucleotides of the portion of exon 2, all of exon 3 through exon 12, and the portion of exon 13 of the human MET gene, or the portion of exon 2, all of exon 3 through exon 12, and the portion of exon 13 of the human MET gene. In some embodiments, the nucleotide sequence at the endogenous non-human animal MET locus that is replaced comprises a nucleotide sequence encoding all or a portion of an extracellular region of an endogenous MET protein. In some embodiments, the nucleotide sequence at the endogenous non-human animal MET locus that is replaced comprises a portion of exon 3, all of exons 4 through 13, and a portion of exon 14 of the endogenous non-human animal (e.g., mouse) MET gene. In some embodiments, after the substitution, the genomic sequence of the endogenous non-human animal (e.g., mouse) MET gene that remains comprises all of exons 1 through 2, part of exon 3, part of exon 14, and all of exons 15 through 22 of the endogenous non-human animal MET gene. In some embodiments, the nucleotide sequence encoding the chimeric MET protein comprises all of non-human