Construction method of a c1r gene humanized animal cell and animal model and application thereof

Abstract

The application belongs to the technical field of animal genetic engineering, and particularly discloses a construction method of C1R gene humanized animal cells and animal models and application thereof. The construction method of the C1R gene humanized animal cells comprises the following steps: introducing a human C1R gene into non-human animal cells, so that the human C1R gene is expressed in the non-human animal cells to produce humanized C1R protein, and meanwhile, the expression of an endogenous C1ra gene in the non-human animal cells is reduced or eliminated. The application preferably adopts CRISPR / Cas9 gene editing technology to accurately insert a genomic sequence encoding a human C1R mature protein into a 2nd exon region of a mouse C1ra gene, while retaining a mouse source promoter, a 5' untranslated region (UTR) and a signal peptide sequence. A mouse cell or model capable of specifically interacting with an anti-human C1R antibody is successfully constructed.

Description

Technical Field

[0001] This invention belongs to the field of animal genetic engineering technology, specifically relating to a method for constructing humanized animal cells and animal models based on the C1R gene and its application. Background Technology

[0002] The complement system is an important component of innate immunity, with activation of the classical pathway beginning with the C1 complex (composed of C1q, C1r, and C1s). The C1R gene encodes the serine protease C1r within the C1 complex, responsible for automatically activating C1s after C1q binds to pathogens or immune complexes, thereby initiating the subsequent complement cascade. Abnormal C1r function is closely associated with various autoimmune diseases, such as systemic lupus erythematosus (SLE), complement deficiency diseases, and autoimmune hemolytic anemia. Existing literature indicates the presence of two C1r genes in mice: C1ra (C1rA) and C1rb (C1rB). C1ra is primarily expressed in the liver, corresponds to the function of human C1R, and is a major functional gene in the classical complement pathway. C1rb is expressed only in the male reproductive system (such as the prostate and coagulation glands), and its function is not closely related to the complement system, but it may be involved in reproductive processes. Therefore, the murine homolog of human C1R is generally considered to be C1ra.

[0003] In order to study the physiological function and pathogenic mechanism of human C1R and evaluate the efficacy of targeted drugs, it is urgent to establish a humanized animal model of C1R at the physiological expression level.

[0004] In preclinical drug research, mice are widely used for efficacy evaluation, pharmacological and toxicological analysis, and other studies. The prerequisite for their application in these studies is that drugs targeting human targets can bind to or activate the corresponding targets in mice. If the test drug cannot bind to the mouse target or activate downstream signaling pathways, mice cannot be used for efficacy, pharmacological, and toxicological evaluations. Comparison of C1R proteins between humans and mice has revealed significant differences in amino acids, with only 80% similarity. This difference in amino acid sequences between human and mouse proteins could prevent drugs targeting human C1R from recognizing mouse C1R targets, especially antibody drugs, where even a single amino acid difference at the recognition site can prevent the antibody from recognizing the target. Therefore, it may be impossible to use wild-type mice to simulate signal transduction against human C1R, complement activation kinetics in human diseases, and the efficacy and toxicity evaluation of human antibody drugs. Animal model experiments are an essential part of preclinical drug research, and the lack of such models could become a bottleneck in drug development. Currently, the lack of human C1R small animal models limits research on human C1R signal transduction, disease research, drug screening, and evaluation. Therefore, constructing humanized mice with the C1R gene has become an urgent need for precision medicine research.

[0005] With the continuous development and maturation of genetic engineering technology, the use of human cells or genes to replace or substitute for endogenous similar cells or genes in animals to establish biological systems or disease models that more closely resemble humans—these humanized animal models—have provided important tools for new clinical treatments and approaches. Among these, gene-humanized animal models utilize gene modification techniques to replace animal homologous genes with normal or mutated human genes, creating gene-humanized animal models in vivo that more closely resemble the characteristics of human diseases. Gene-humanized animals not only have significant application value—for example, improving drug targeting in vivo—but more importantly, due to the presence of human gene fragments, the animals can express or partially express proteins with human functions, thereby greatly reducing the discrepancy between preclinical animal experiments and clinical trials, improving model accuracy, and providing possibilities for further exploration of the mysteries of human diseases, development of novel treatment methods, and preclinical in vivo drug screening and validation. Summary of the Invention

[0006] In view of this, the present invention provides a method for constructing and applying humanized animal cells and animal models of the C1R gene; the present invention introduces the human C1R gene into non-human animal cells, so that the human C1R gene is expressed in the non-human animal cells to produce humanized C1R protein, while reducing or eliminating the expression of endogenous C1ra gene in the non-human animal cells.

[0007] The technical solution provided by this invention is as follows:

[0008] In a first aspect, the present invention provides a method for constructing humanized animal cells with the C1R gene, comprising:

[0009] The human C1R gene is introduced into non-human animal cells, causing the human C1R gene to be expressed in the non-human animal cells to produce humanized C1R protein, while reducing or eliminating the expression of endogenous C1ra gene in the non-human animal cells.

[0010] The C1ra gene mRNA sequence is shown in SEQ ID NO.32, and the C1ra amino acid sequence is shown in SEQ ID NO.33.

[0011] In some embodiments, gene editing technology is used to construct a humanized animal model of the C1R gene, forming a humanized C1R gene in the non-human animal cells.

[0012] Optionally, the non-human animal is a rodent.

[0013] Optionally, the non-human animal cell is a fertilized egg cell.

[0014] Optionally, the rodent is a mouse.

[0015] The construction method involves knocking the DNA sequence (SEQ ID NO. 6) of the human C1R translated mature protein into exon 2 of the mouse C1ra gene, while retaining the mouse promoter, 5'UTR, and signal peptide sequence.

[0016] The overall strategy for humanization in this invention is as follows: At the DNA level, the DNA sequence (nucleic acid sequence as shown in SEQ ID NO. 6, corresponding amino acid sequence as shown in SEQ ID NO. 17) encoding the mature protein of the human C1R gene (ENSEMBL Gene ID: ENSG00000159403) transcript (ENSEMBL Gene ID: ENST00000647956.2) is knocked into exon 2 of the mouse C1ra gene (ENSEMBL Gene ID: ENSMUSG00000055172) transcript (ENSMUST00000068593.9). Simultaneously, the mouse promoter, 5' untranslated region (UTR) (SEQ ID NO. 29), and signal peptide sequence (SEQ ID NO. 30) are retained, and the corresponding amino acid sequence of the signal peptide is shown in SEQ ID NO. 31.

[0017] The humanized C1R gene is selected from at least one of the following groups:

[0018] (1) The CDS coding sequence of the gene is shown in SEQ ID NO.1;

[0019] (2) The mRNA sequence transcribed from the gene is shown in SEQ ID NO.2;

[0020] (3) The protein sequence encoded by the gene is shown in SEQ ID NO.3.

[0021] As one embodiment of the present invention, the construction method includes: providing a mixture of a human C1R gene homologous recombination vector, sgRNA and Cas9, wherein the Cas9 includes Cas9 mRNA and / or Cas9 protein; injecting the mixture into the non-human animal cells; culturing the non-human animal cells to obtain the C1R gene humanized animal cells.

[0022] The human C1R gene homologous recombination vector includes a 5' homologous arm, a human C1R gene fragment, and a 3' homologous arm arranged sequentially from the 5' end to the 3' end.

[0023] The 5' homologous arm is a DNA fragment homologous to the 5' end of exon 2 of the mouse C1R gene, and the 3' homologous arm is a DNA fragment homologous to the 3' end of exon 2 of the mouse C1R gene.

[0024] The DNA sequence of the 5' homologous arm is shown in SEQ ID NO.4, and the DNA sequence of the 3' homologous arm is shown in SEQ ID NO.5.

[0025] The target site of the sgRNA in the mouse C1R gene is located in exon 2 of the mouse C1R gene.

[0026] The sequence targeted by the sgRNA is shown in SEQ ID NO.7.

[0027] The human C1R gene fragment is shown in SEQ ID NO.18.

[0028] The human C1R gene fragment (SEQ ID NO.18) is the 58th to 2118th nucleotides (SEQ ID NO.6) of the coding sequence (SEQ ID NO.1) of the human C1R gene (ENSEMBL Gene ID: ENSG00000159403) transcript (ENSEMBL ID: ENST00000647956.2) and the transcription termination signal bGH poly(A) signal.

[0029] Secondly, the present invention provides a method for constructing a humanized animal model of the C1R gene, the method comprising implanting humanized animal cells of the C1R gene constructed by the method described above or embryos developed from them into a surrogate mother for continued development, thereby obtaining F0 generation positive animals.

[0030] Optionally, the non-human animal is a rodent.

[0031] Optionally, the rodent is a mouse.

[0032] Optionally, this construction method also includes using F0 generation positive mice for hybridization and self-pollination to expand the population size, and breeding homozygous mice through self-pollination of heterozygous mice, thereby establishing a stable genetic C1R gene humanized mouse strain.

[0033] Thirdly, the present invention provides the application of a C1R gene humanized animal model constructed by the method described above, or a C1R gene humanized animal obtained by the method described above for constructing C1R gene humanized animal cells, in screening or evaluating drugs acting on human C1R protein.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] We have innovatively proposed a strategy and application scheme for constructing humanized animal cells and models of the C1R gene. This model has greatly promoted the research process related to the human C1R gene or protein, especially demonstrating great potential in evaluating the efficacy of drugs targeting human C1R, and providing valuable guidance for predicting the clinical efficacy of drug development. Furthermore, this model plays a crucial role in drug safety evaluation, serving as an ideal platform for in-depth in vivo toxicological evaluation of human C1R-targeted drugs in the preclinical stage, thereby ensuring the safety and efficacy of drug development.

[0036] Preferably, this invention ingeniously utilizes CRISPR / Cas9 gene editing technology to precisely perform an innovative modification in mice with intact immune systems: the genomic sequence encoding the mature human C1R protein is accurately inserted into the exon2 position of the mouse C1ra gene, thereby successfully constructing a mouse model capable of specifically recognizing and binding to anti-human C1R antibodies. Compared to traditional mouse models, this innovative model not only achieves the humanized replacement of key target molecules but also provides an unprecedented platform for drug screening and evaluation targeting the human C1R gene while preserving the mouse's complete immune function, making it an ideal choice for preclinical drug development.

[0037] Furthermore, the C1R humanized mouse model created in this invention ingeniously integrates a mouse promoter, a 5'UTR region, and a signal peptide sequence. This design ensures the accurate expression and localization of the mature human protein in mice. Through this sophisticated construction, we obtained a mouse model carrying the mature human C1R protein. This model can not only be effectively used for screening human C1R-targeted drugs, but also, thanks to the addition of mouse regulatory elements, ensures the normal functional expression and accurate localization of these human proteins in the mouse physiological environment. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or existing methods and experiments, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 Schematic diagram of the mouse C1ra gene and the human C1R gene;

[0040] Figure 2 This is a schematic diagram of the C1R gene structure in the modified humanized mouse.

[0041] Figure 3 A schematic diagram of a recombination strategy for humanizing the mouse C1ra gene;

[0042] Figure 4 Electrophoresis image for enzyme digestion identification of homologous recombination vector;

[0043] Figure 5 Electrophoresis images of homologous recombination PCR identification results for F0 and F1 generation mice; where A is the electrophoresis image of homologous recombination identification of the 5' homologous arm of F0 generation mice, B is the electrophoresis image of homologous recombination identification of the 3' homologous arm of F0 generation mice; C is the 5' homologous arm of F1 generation mice, and D is the electrophoresis image of homologous recombination identification of the 3' homologous arm; the lane numbers correspond to the mouse numbers; WT is the wild-type control; M is the 1kb DNA marker;

[0044] Figure 6 A schematic diagram illustrating the design of qPCR primer positions;

[0045] Figure 7 The results are qPCR results for F2 generation wild-type and homozygous mice; HO represents hC1R homozygous mice; WT represents wild-type mice; M represents a 100bp DNA marker.

[0046] Figure 8 A represents the expression results of human C1R protein in the serum of wild-type mice and homozygous mice; B represents the expression results of human C1R protein in the serum; HO represents homozygous mice; WT represents wild-type mice.

[0047] Figure 9 The spectrum of PBR322-MCS. Detailed Implementation

[0048] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0049] The mouse strains, biochemical reagents, and experimental instruments used in the embodiments of this invention are as follows:

[0050] C57BL / 6 mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0051] EcoRI restriction endonuclease was purchased from NEB, catalog number R3101M;

[0052] The In-Fusion HD Cloning Kits were purchased from Takara, item number 639650.

[0053] The sgRNA in vitro transcription kit was purchased from Ambion, catalog number AM1354;

[0054] Escherichia coli DH5α competent cells were purchased from TaKaRa, catalog number 9057;

[0055] Cas9 mRNA sourced from SIGMA, catalog number CAS9MRNA-1EA;

[0056] The Human C1R ELISA Kit was purchased from Abcam, product number ab170245;

[0057] The Mouse C1R Elisa Kit was purchased from Boster Bio, product number EK1955;

[0058] T7 endonuclease I detection kit, purchased from NEB, catalog number M0302S;

[0059] The PCR product gel extraction kit was the QIAquick Gel Extraction Kit, purchased from QIAGEN, catalog number 28706.

[0060] PBR322-MCS (such as) Figure 9 ).

[0061] Example 1: Sequence Design

[0062] The human C1R gene contains multiple transcripts. The sequence design in this embodiment is mainly based on one of the transcripts.

[0063] The overall humanization strategy is as follows: At the DNA level, the DNA sequence encoding the mature protein of the human C1R gene (ENSEMBL Gene ID: ENSG00000159403) transcript (ENSEMBL Gene ID: ENST00000647956.2) is knocked into exon 2 of the mouse C1ra gene (ENSEMBL Gene ID: ENSMUSG00000055172) transcript (ENSMUST00000068593.9). Simultaneously, the mouse promoter, the 5' untranslated region (UTR) (SEQ ID NO.29), and the signal peptide sequence (SEQ ID NO.30) are preserved. The amino acid sequence corresponding to the signal peptide is shown in SEQ ID NO.31.

[0064] The coding sequence of the human C1R gene (ENSEMBL Gene ID: ENSG00000159403) transcript (ENSEMBL ID: ENST00000647956.2) knocked in is shown in SEQ ID NO.6, and the corresponding protein amino acid sequence is shown in SEQ ID NO.17.

[0065] A schematic diagram comparing the structures of the mouse C1ra gene (ENSEMBL Gene ID: ENSMUSG00000055172) and the human C1R gene is shown below. Figure 1 The final schematic diagram of the modified humanized mouse C1R gene structure is shown below. Figure 2 .

[0066] Example 2: Design and Construction of Recombinant Vector PBR322-C1R

[0067] Based on the sequence design in Example 1, further designs were made as follows: Figure 3 The targeting scheme is shown. The vector structure includes a 5' homologous arm, a human C1R gene fragment (including the human C1R gene coding sequence and the transcription termination signal bGH poly(A) signal), and a 3' homologous arm. Among them:

[0068] The 5' homologous arm (SEQ ID NO.4) is nucleotides 124487663-124490662 of the transcript (ENSMUST00000068593.9) of the mouse C1ra gene (ENSEMBL Gene ID: ENSMUSG00000055172);

[0069] The 3' homologous arm (SEQ ID NO.5) is nucleotides 124490663-124493662 of the transcript (ENSMUST00000068593.9) of the mouse C1ra gene (ENSEMBL Gene ID: ENSMUSG00000055172);

[0070] The human C1R gene fragment (SEQ ID NO.18) is the 58th to 2118th nucleotides (SEQ ID NO.6) of the coding sequence (SEQ ID NO.1) of the human C1R gene (ENSEMBL Gene ID: ENSG00000159403) transcript (ENSEMBL ID: ENST00000647956.2) and the transcription termination signal bGH poly(A) signal.

[0071] According to the design scheme, the mRNA sequence of C1R expressed in the modified humanized mice is shown in SEQ ID NO.19, and the protein sequence is shown in SEQ ID NO.20. The C1R humanized mouse model was constructed using the CRISPR / Cas9 protocol, as per the design strategy.

[0072] Figure 3 This is a schematic diagram of a recombination strategy for humanizing the mouse C1ra gene.

[0073] The construction process of the vector (PBR322-C1R) is as follows:

[0074] Using C57BL / 6 mouse genomic DNA (or a BAC library as a template), PCR amplification was performed to obtain the 5' and 3' homologous arms. Using a synthesized human C1R gene fragment as a template, PCR amplification was performed to obtain the human C1R gene fragment. The fragment was ligated into the PBR322-MCS plasmid using an in-fusion kit to obtain the homologous recombination vector PBR322-C1R. After verification of correctness by enzyme digestion and sequencing, the vector was used for subsequent microinjection of fertilized eggs. The ligation order of each fragment was linearly inserted into the multiple cloning site according to the relationship: 5' homologous arm → human C1R gene fragment → 3' homologous arm.

[0075] Validation of vector PBR322-C1R:

[0076] Five PBR322-C1R clones were randomly selected and digested with the restriction endonuclease Sca I for verification. Electrophoresis of the digestion products should show fragments of sizes 7584 bp, 3047 bp, 1547 bp, and 540 bp. See the digestion results below. Figure 4 The plasmid digestion results were as expected, indicating that the plasmid digestion verification results were correct. The plasmid was also verified to be correct by sequencing by a sequencing company, and subsequent experiments were conducted.

[0077] Example 3: C1ra gene sgRNA design

[0078] The target sequence of sgRNA determines its targeting specificity and the efficiency with which it induces Cas9 cleavage of the target gene. Therefore, efficient and specific target sequence selection and design are prerequisites for constructing sgRNA homologous recombination vectors.

[0079] Based on the targeting protocol, sgRNA sequences that recognize target sites (sgRNA1-sgRNA10) were designed and synthesized.

[0080] Based on the function and sequence characteristics of the mouse C1ra gene, the 50 bp upstream and 150 bp downstream of the knock-in site were selected as gRNA target sequences. From the gRNA design website, gRNAs with an MIT Specificity Score > 50 were selected as candidate gRNAs. The target site sequences of each sgRNA on the C1ra gene are as follows:

[0081] sgRNA-1 target site sequence (SEQ ID NO.7): 5'-GAGCTTCTGAGGGAGGTAAA TGG-3'

[0082] sgRNA-2 target site sequence (SEQ ID NO.8): 5'-GGTCTTCTGGCAGTTTGACGTGG-3'

[0083] sgRNA-3 target site sequence (SEQ ID NO.9): 5'-GCTTCACCCTGTACCCCATGGGG-3'

[0084] sgRNA-4 target site sequence (SEQ ID NO.10): 5'-GCCTTCCACCCCATAGAACAGGG-3'

[0085] sgRNA-5 target site sequence (SEQ ID NO.11): 5'-AGCTTCACCCTGTACCCCATGGG-3'

[0086] sgRNA-6 target site sequence (SEQ ID NO.12): 5'-TCACTGTCCCCATGGGGTACAGG-3'

[0087] sgRNA-7 target site sequence (SEQ ID NO.13): 5'-CCCCATAGAACAGGGTCACCAGG-3'

[0088] sgRNA-8 target site sequence (SEQ ID NO.14): 5'-CCTGGTGACCCTGTTCTATGGGG-3'

[0089] sgRNA-9 target site sequence (SEQ ID NO.15): 5'-GGTGACCCTGTTCTATGGGGTGG-3'

[0090] sgRNA-10 target site sequence (SEQ ID NO.16): 5'-CCCTCAGAAGCTCTATGGAGAGG-3'

[0091] Analysis revealed that only sgRNA-1 met the MIT Specificity Score > 50. Therefore, sgRNA-1 (SEQ ID NO. 7) was selected as the preferred gRNA for subsequent construction.

[0092] Example 4: Construction of a C1R gene-humanized mouse model

[0093] 1. Intrauterine injection of fertilized eggs and embryo transfer

[0094] Fertilized eggs from C57BL / 6 mice were collected, and the PBR322-C1R recombinant vector, Cas9 mRNA, and gRNA (sgRNA-1, sequence SEQ ID NO.7) were mixed according to the method in the "Mouse Embryo Manipulation Experiment Manual (Third Edition)". The fertilized eggs were then microinjected. After a short period of incubation, the injected fertilized eggs were transplanted into the oviduct of the recipient mother mouse to obtain genetically modified humanized mice, which were then called founder mice (F0 generation).

[0095] 2. Identification of C1R humanized mice

[0096] After the F0 generation mice were born, their tails were clipped and their genomes extracted. Long-fragment PCR was used to identify positive clones for homologous recombination at the 5' and 3' homologous arms, respectively. The primer positions for PCR identification are shown in the diagram. Figure 3 As shown, the specific identification information for homologous recombination in F0 generation and subsequent F1 generation mice is as follows:

[0097] The criteria for identifying 5'-arm homologous recombination are as follows:

[0098] Primer sequence information is as follows:

[0099]

[0100] The PCR reaction system is as follows:

[0101]

[0102] PrimeStar GXL (GXL PCR Buffer): TaKaRa, Code No: R050A

[0103] The PCR reaction procedure is as follows:

[0104]

[0105] The criteria for identifying homologous recombination in the 3' arm are as follows:

[0106] Primer sequence information is as follows:

[0107]

[0108] The PCR reaction system is as follows:

[0109]

[0110] PrimeStar GXL (GXL PCR Buffer): TaKaRa, Code No: R050A.

[0111] The PCR reaction procedure is as follows:

[0112]

[0113] According to the design strategy and identification protocol, a 5.5 kb fragment should be amplified from the 5' arm homologous recombination-positive genome, and a 7.2 kb fragment from the negative genome; a 3.3 kb fragment should be amplified from the 3' arm homologous recombination-positive genome, and a 6.6 kb fragment from the negative genome. The identification electrophoresis results are as follows: Figure 5 China A and Figure 5 As shown in Figure B. After PCR identification and sequencing confirmation, three F0 generation positive mice were obtained, numbered 2, 3, and 17.

[0114] Adult F0 mice were mated with wild-type C57BL / 6 mice to obtain F1 mice. After birth, the tails of F1 mice were clipped and their genomes extracted. Using the same conditions as for identifying homologous recombination in F0 mice, PCR was performed targeting both the 5' and 3' homologous arms to confirm whether the obtained F1 mice were offspring of correctly homologously recombinated C1R gene-humanized mice. Positive mice were identified as F1 heterozygous mice. PCR results are shown below. Figure 5 C and Figure 5 As shown in Figure D, the positive mice are mice 3, 8, 10, 11, 12, 14, 16, and 17. C1R humanized homozygous mice were obtained from the F1 generation heterozygous mice through self-crossing; a stable hC1R humanized mouse strain was established.

[0115] The above results demonstrate that this method can be used to efficiently construct and obtain a stable, passageable humanized mouse model of the C1R gene.

[0116] Example 5: Analysis of C1R mRNA Expression in Humanized Mice

[0117] Total RNA was extracted from the livers of a 6-week-old C1R humanized homozygous mouse and a 6-week-old littermate wild-type mouse. The RNA was reverse transcribed into cDNA using a reverse transcription kit (TransGen Biotech, AT311). The cDNA was then used as a template for PCR to verify the expression level of human C1R mRNA in the humanized mouse.

[0118] Primers were designed for the detection of murine C1ra mRNA (SEQ ID NO.32) and humanized C1R mRNA (SEQ ID NO.19) in this experiment, as shown in the figure. Figure 6 The positions of primers P5 / P6 and P7 / P8 are shown in the figure. The primer sequences for detecting mouse C1ra mRNA expression are as follows:

[0119] P5: 5' - ggtcttctggcagtttgacg-3' (SEQ ID NO.25), and

[0120] P6: 5'-AGACATAGCCTGAGGGCTCT-3' (SEQ ID NO.26), PCR product is 300bp;

[0121] The primer sequences for detecting human C1R mRNA expression are as follows:

[0122] P7: 5'-TCCTGGTGACCCTGTTCTAT-3' (SEQ ID NO.27), and

[0123] P8: 5'-GGTCAAACTGCTGGAAGACG-3' (SEQ ID NO.28), PCR product size is 314bp.

[0124] The PCR reaction system and conditions for both primer pairs P5 / P6 and P7 / P8 are the same: 20 μL PCR system, reaction conditions: 94℃, 4 min; (94℃, 15 sec; 58℃, 15 sec; 72℃, 45 sec, 32 cycles); 72℃, 5 min; 12℃ incubation.

[0125] RNA detection results as follows Figure 7 As shown in the figure; the results indicate that active human C1R expression can be detected at the mRNA level in C1R humanized mice.

[0126] Example 6: Analysis of C1R protein expression levels in humanized mice

[0127] To verify whether human C1R protein (SEQ ID NO.3) was expressed after humanization, the expression of C1R protein in the serum of wild-type mice and C1R humanized homozygous mice was detected by ELISA. The results were consistent with expectations. Figure 8 , Figure 8 In Figure A, the expression results of mouse-derived C1R protein in serum are shown. Figure 8(B in the table shows the results of human C1R protein expression in serum): Wild-type mice expressed murine C1R protein (SEQ ID NO.33) but did not express human C1R protein. C1R humanized homozygous mice expressed human C1R protein but did not express murine C1R protein.

[0128] These results demonstrate that human C1R protein can be expressed in C1R-modified mice, and this mouse model can be used for preclinical in vivo drug screening and validation and other related studies.

[0129] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that these are merely illustrative examples, and any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing humanized animal cells with the C1R gene, characterized in that, include: Provides a mixture of a human C1R gene homologous recombination vector, sgRNA, and Cas9, wherein the Cas9 is Cas9 mRNA and / or Cas9 protein; The mixture was injected into the non-human animal cells; The injected non-human animal cells were cultured to obtain humanized animal cells with the C1R gene. The construction method involves knocking the DNA sequence encoding the mature human C1R protein into exon 2 of the mouse C1ra gene while retaining the mouse promoter, 5' untranslated region, and signal peptide sequence. This introduces the human C1R gene into non-human animal cells, causing the human C1R gene to be expressed in the non-human animal cells to produce humanized C1R protein, while reducing or eliminating the expression of the endogenous C1ra gene in the non-human animal cells. The DNA sequence encoding the mature human C1R protein is shown in SEQ ID NO. 6; The human C1R gene homologous recombination vector includes a 5' homologous arm, a human C1R gene fragment, and a 3' homologous arm arranged sequentially from the 5' end to the 3' end; The 5' homologous arm is a DNA fragment homologous to the 5' end of exon 2 of the mouse C1ra gene, and its sequence is shown in SEQ ID NO. 4; The 3' homologous arm is a DNA fragment homologous to the 3' end of exon 2 of the mouse C1ra gene, and its sequence is shown in SEQ ID NO. 5; The human C1R gene fragment is shown in SEQ ID NO.18; The sgRNA targets the sequence in exon 2 of the mouse C1ra gene; the sequence targeted by the sgRNA is shown in SEQ ID NO.7; the non-human animal cell is a mouse zygote.

2. A method for constructing a humanized animal model of the C1R gene, characterized in that, The construction method includes implanting humanized animal cells with the C1R gene obtained by the method described in claim 1, or embryos developed from them, into a surrogate mother for continued development, thereby obtaining F0 generation positive animals.

3. The application of C1R gene humanized animal cells constructed by the method for constructing C1R gene humanized animal cells as described in claim 1, or C1R gene humanized animal models constructed by the method for constructing C1R gene humanized animal models as described in claim 2, in screening or evaluating drugs acting on human C1R protein.

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