A kind of RHO-like protein and its application in constructing retinopathy animal model
By performing humanized substitution in the second exon region of the mouse Rho gene and CRISPR/Cas9-mediated site knock-in, animal models carrying or not carrying the RHO pathogenic mutation were constructed, solving the problem of inaccurate models in existing technologies and achieving efficient evaluation of gene editing tools and accurate simulation of retinal disease research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI FIRST PEOPLES HOSPITAL
- Filing Date
- 2026-04-14
- Publication Date
- 2026-06-23
AI Technical Summary
Existing technologies make it difficult to establish a retinal lesion model in mice that is consistent with human RHO gene mutations. This leads to inaccurate assessment of the editing efficiency and editing window of gene editing tools in the context of human sequences, thus limiting the development and validation of gene editing technologies.
Humanized replacements were performed in the second exon region of the endogenous Rho gene in mice using gene knock-in technology to construct animal models carrying or not carrying the pathogenic Rho mutation. Precise replacement and mutation introduction were achieved through CRISPR/Cas9-mediated site-directed knock-in, and the stability and accuracy of the models were ensured by combining homologous arm PCR and sequencing verification.
It provides a comparative model of control and mutant groups under the same genetic background, which can accurately assess the editing efficiency and specificity of gene editing tools, improve the genetic stability and phenotypic interpretability of the model, and support the study of retinal disease mechanisms and drug screening.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of animal models, specifically relating to an RHO-like protein and its application in constructing animal models of retinal diseases. Background Technology
[0002] In the rod cells of the retina, opsin combines with 11-cis-retinal, formed from the oxidation of vitamin A, to form rhodopsin (RHO). Under light, 11-cis-retinal is converted to all-trans-retinal, which is the first step in visual signal transduction. RHO Gene mutations are one of the most common causes of autosomal dominant retinitis pigmentosa (RP).
[0003] Of particular note is that RHO c.403 C>T (p.R135W) is a relatively common and clearly pathogenic mutation in the population. Patients carrying this mutation experience rapid disease progression and poor visual prognosis, suggesting a high clinical severity.
[0004] To gain a deeper understanding of the pathogenic mechanism of this mutation and develop targeted therapies, there is an urgent need for animal models that can accurately simulate the pathological process of human mutations. Summary of the Invention
[0005] This invention provides a humanized mouse model without the RHO pathogenic mutation (which can be used as a control model) and a retinal lesion model carrying the RHO pathogenic mutation, thereby providing a preclinical research platform that highly simulates human diseases for studying the mechanism of retinal degeneration caused by this mutation and verifying the effectiveness and safety of gene therapy strategies.
[0006] In a first aspect of the present invention, a humanized RHO recombinant protein is provided, wherein the humanized RHO recombinant protein is a chimeric protein obtained by replacing the amino acid region encoded by the second exon in a non-human mammalian Rho protein with the amino acid region encoded by the second exon in a human RHO protein.
[0007] In another preferred embodiment, the animal includes: a mouse, a rat, a pig, a dog, a rabbit, or a non-human primate.
[0008] In another preferred embodiment, the animal is a rodent.
[0009] In another preferred embodiment, the animal is a mouse.
[0010] In another preferred embodiment, the recombinant protein is an isolated or purified recombinant protein.
[0011] In another preferred embodiment, there was no significant difference in the expression level A1 of the humanized RHO recombinant protein in the non-human mammal and the expression level A0 of the wild-type Rho protein in the non-human mammal.
[0012] In another preferred embodiment, the lack of significant difference means 0.8 ≤ A1 / A0 ≤ 1.2, and more preferably 0.9 ≤ A1 / A0 ≤ 1.1.
[0013] In another preferred embodiment, the humanized RHO recombinant protein is a humanized RHO recombinant protein used to construct a negative control animal model of retinopathy.
[0014] In another preferred embodiment, the recombinant protein comprises the sequence shown in SEQ ID NO:1.
[0015] In another preferred embodiment, the RHO recombinant protein further comprises: a retinal pathogenic mutation.
[0016] In another preferred embodiment, the retinal pathogenic mutations include: R135W, R135L, P171L, A164V, and L125R; The amino acid numbering of the mutation is based on the human RHO protein, whose Genbank accession number is NP_000530.1.
[0017] In a second aspect of the invention, an RHO recombinant protein mutant is provided, said mutant being an RHO recombinant protein mutant having one or more site mutations selected from the group consisting of: R135W, R135L, P171L, A164V, L125R; The amino acid numbering of the mutation is based on the human RHO protein, whose Genbank accession number is NP_000530.1.
[0018] In another preferred embodiment, the RHO recombinant protein mutant is an isolated or purified RHO recombinant protein mutant.
[0019] In another preferred embodiment, the RHO recombinant protein mutant comprises the amino acid sequence shown in SEQ ID NO:2.
[0020] In a third aspect of the invention, a polynucleotide is provided that encodes the RHO recombinant protein described in the first aspect of the invention.
[0021] In another preferred embodiment, the polynucleotide is selected from the group consisting of DNA, RNA, cDNA, or combinations thereof.
[0022] In another preferred embodiment, the polynucleotide is a separate polynucleotide.
[0023] In a fourth aspect of the invention, a polynucleotide is provided that encodes the RHO recombinant protein mutant described in the second aspect of the invention.
[0024] In another preferred embodiment, the polynucleotide is selected from the group consisting of DNA, RNA, cDNA, or combinations thereof.
[0025] In another preferred embodiment, the polynucleotide is a separate polynucleotide.
[0026] In a fifth aspect of the invention, a carrier is provided that comprises the polynucleotide described in the third aspect of the invention.
[0027] In another preferred embodiment, the vector includes bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
[0028] In another preferred embodiment, the carrier is an expression carrier.
[0029] In another preferred embodiment, the vector is the pET28a plasmid.
[0030] In a sixth aspect of the invention, a carrier is provided that comprises the polynucleotide described in the fourth aspect of the invention.
[0031] In another preferred embodiment, the vector includes bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
[0032] In another preferred embodiment, the carrier is an expression carrier.
[0033] In another preferred embodiment, the vector is the pET28a plasmid.
[0034] In a seventh aspect of the invention, a host cell is provided, said host cell containing the vector described in the fifth aspect of the invention, or said host cell having the polynucleotide described in the third aspect of the invention integrated into its genome.
[0035] In another preferred embodiment, suitable host cells include Gram-positive bacteria, Gram-negative bacteria, actinomycetes, yeasts, and fungi. The Gram-positive bacteria include, but are not limited to, Bacillus subtilis; the Gram-negative bacteria include, but are not limited to, Escherichia coli; the actinomycetes include, but are not limited to, Streptomyces; the yeasts include, but are not limited to, Saccharomyces cerevisiae; and the fungi include, but are not limited to, Aspergillus. Their cells are all commonly used host cells for recombinant vectors.
[0036] In another preferred embodiment, the host cell further contains a vector for expressing the RHO recombinant protein.
[0037] In another preferred embodiment, the host cell expresses the RHO recombinant protein.
[0038] In another preferred embodiment, the host cell will not generate an animal or plant body.
[0039] In an eighth aspect of the invention, a host cell is provided, said host cell containing the vector described in the sixth aspect of the invention, or said host cell having the polynucleotide described in the fourth aspect of the invention integrated into its genome.
[0040] In another preferred embodiment, suitable host cells include Gram-positive bacteria, Gram-negative bacteria, actinomycetes, yeasts, and fungi. The Gram-positive bacteria include, but are not limited to, Bacillus subtilis; the Gram-negative bacteria include, but are not limited to, Escherichia coli; the actinomycetes include, but are not limited to, Streptomyces; the yeasts include, but are not limited to, Saccharomyces cerevisiae; and the fungi include, but are not limited to, Aspergillus. Their cells are all commonly used host cells for recombinant vectors.
[0041] In another preferred embodiment, the host cell further contains a vector for expressing the RHO recombinant protein mutant.
[0042] In another preferred embodiment, the host cell expresses the RHO recombinant protein mutant.
[0043] In another preferred embodiment, the host cell will not generate an animal or plant body.
[0044] In a ninth aspect of the present invention, a method for preparing the RHO recombinant protein described in the first aspect of the present invention is provided, comprising the steps of: (i) Under suitable expression conditions, host cells as described in the seventh aspect of the present invention are cultured to express the RHO recombinant protein as described in the first aspect of the present invention; (ii) Isolate the expression product to obtain the RHO recombinant protein.
[0045] In a tenth aspect of the present invention, a method for preparing the RHO recombinant protein mutant described in the second aspect of the present invention is provided, comprising the steps of: (i) Under suitable expression conditions, host cells as described in the eighth aspect of the present invention are cultured to express the RHO recombinant protein mutant as described in the second aspect of the present invention; (ii) Isolate the expression product to obtain the RHO recombinant protein mutant.
[0046] In the eleventh aspect of the present invention, the use of the polynucleotide described in the third or fourth aspect of the present invention, or the vector described in the fifth or sixth aspect of the present invention, or the host cell described in the seventh or eighth aspect of the present invention, is provided for the preparation of non-human animal models.
[0047] In another preferred embodiment, the non-human animal model is used for scientific research on retinal diseases and / or for screening drugs for retinal diseases.
[0048] In another preferred embodiment, the non-human animal model is a retinal lesion model with a retinal pathogenic mutation.
[0049] In another preferred embodiment, the non-human animal model is a negative control model in which no retinal pathogenic mutations are introduced.
[0050] In a twelfth aspect of the present invention, a method for preparing a non-human animal model is provided, comprising the steps of: (S1) Provides modified non-human animal pluripotent stem cells expressing the RHO recombinant protein described in the first aspect of the invention; and (S2) Use the modified non-human animal pluripotent stem cells described in (S1) to generate non-human animal models.
[0051] In another preferred embodiment, the method is a method for non-diagnostic and non-therapeutic purposes.
[0052] In another preferred embodiment, the pluripotent stem cell is a fertilized egg.
[0053] In another preferred embodiment, the pluripotent stem cell is an early blastomeres.
[0054] In another preferred embodiment, the early stage includes: a 2-cell stage, a 4-cell stage, or an 8-cell stage.
[0055] In another preferred embodiment, the pluripotent stem cells are pluripotent-like stem cells obtained through artificial induction.
[0056] In another preferred embodiment, the pluripotent stem cells are selected from the group consisting of extended pluripotent stem cells (EPS) and pluripotent stem cells (TPS).
[0057] In another preferred embodiment, the non-human animal pluripotent stem cells contain the polynucleotides described in the third aspect of the present invention.
[0058] In another preferred embodiment, the non-human animal model is a humanized non-human animal model.
[0059] In another preferred embodiment, the humanized non-human animal model is a negative control model that has not introduced pathogenic retinal mutations.
[0060] In another preferred embodiment, the method includes: (S1') Provides modified non-human animal pluripotent stem cells expressing the RHO recombinant protein described in the first aspect of the present invention; (S2') Use the modified non-human animal pluripotent stem cells described in (S1') to generate humanized non-human animal models.
[0061] In another preferred embodiment, in step (S1), the pluripotent stem cells contain the sequence shown in SEQ ID NO:10.
[0062] In another preferred embodiment, the method includes: (S1') Provides modified non-human animal pluripotent stem cells expressing the RHO recombinant protein described in the first aspect of the present invention; (S2') Using the modified non-human animal pluripotent stem cells described in (S1') to generate heterozygous humanized non-human animal models; and (S3') The hybrid humanized non-human animal model is mated with a wild-type non-human animal to obtain a homozygous humanized non-human animal model.
[0063] In another preferred embodiment, step (S1) further includes the following sub-steps: (S1-1) Provides non-human animal pluripotent stem cells; (S1-2) Humanization of non-human animal pluripotent stem cells is performed using sgRNA, Cas9 mRNA, and donor sequence to provide modified non-human animal pluripotent stem cells expressing the RHO recombinant protein described in the first aspect of the present invention. The donor sequence from 5'-3' includes the structure shown in Formula I: X1-X2-X3(I) in, X1 is a 5' homologous arm; X2 is the wild-type human exon 2; X3 is a 3' homologous arm.
[0064] In another preferred embodiment, the donor sequence is the sequence shown in SEQ ID NO:7.
[0065] In another preferred embodiment, the sgRNA comprises the sequences shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
[0066] In a thirteenth aspect of the present invention, a method for preparing a non-human animal model is provided, comprising the steps of: (S1) Provides modified non-human animal pluripotent stem cells expressing the RHO recombinant protein mutant described in the second aspect of the invention; and (S2) Use the modified non-human animal pluripotent stem cells described in (S1) to generate non-human animal models.
[0067] In another preferred embodiment, the method is a method for non-diagnostic and non-therapeutic purposes.
[0068] In another preferred embodiment, the pluripotent stem cell is a fertilized egg.
[0069] In another preferred embodiment, the pluripotent stem cell is an early blastomeres.
[0070] In another preferred embodiment, the early stage includes: a 2-cell stage, a 4-cell stage, or an 8-cell stage.
[0071] In another preferred embodiment, the pluripotent stem cells are pluripotent-like stem cells obtained through artificial induction.
[0072] In another preferred embodiment, the pluripotent stem cells are selected from the group consisting of extended pluripotent stem cells (EPS) and pluripotent stem cells (TPS).
[0073] In another preferred embodiment, the non-human animal pluripotent stem cells contain the polynucleotides described in the fourth aspect of the present invention.
[0074] In another preferred embodiment, the non-human animal model is a humanized non-human animal model.
[0075] In another preferred embodiment, the humanized non-human animal model is a negative control model that has not introduced pathogenic retinal mutations.
[0076] In another preferred embodiment, the method includes (S1'') Provides modified non-human animal pluripotent stem cells expressing the RHO recombinant protein mutant described in the third aspect of the present invention; (S2'') Use the modified non-human animal pluripotent stem cells described in (S1'') to generate humanized non-human animal models with retinal pathogenic mutations.
[0077] In another preferred embodiment, in step (S1), the pluripotent stem cells contain the sequence shown in SEQ ID NO:17.
[0078] In another preferred embodiment, the method includes: (S1'') Provides modified non-human animal pluripotent stem cells expressing the RHO recombinant protein mutant described in the third aspect of the present invention; (S2'') Using the modified non-human animal pluripotent stem cells described in (S1'') to generate non-human animal models; and (S3'') The non-human animal model obtained in step (S2'') is mated with a homozygous humanized non-human animal model to obtain a humanized non-human animal model with pathogenic mutations.
[0079] In another preferred embodiment, the homozygous humanized non-human animal model refers to the homozygous humanized non-human animal model obtained by the method of the twelfth aspect of the present invention.
[0080] In another preferred embodiment, step (S1) further includes the following sub-steps: (S1-1) Provides non-human animal pluripotent stem cells; (S1-2) Humanization of non-human animal pluripotent stem cells is performed using sgRNA, Cas9 mRNA, and donor sequence to provide modified non-human animal pluripotent stem cells expressing the RHO recombinant protein mutant described in the second aspect of the present invention. The donor sequence from 5'-3' includes the structure shown in Formula I: X1-X2-X3(I) in, X1 is a 5' homologous arm; X2 is the human exon 2 that introduces the pathogenic mutation; X3 is a 3' homologous arm.
[0081] In another preferred embodiment, the donor sequence is the sequence shown in SEQ ID NO:14.
[0082] In another preferred embodiment, the sgRNA comprises the sequences shown in SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:5 and SEQ ID NO:13.
[0083] In a fourteenth aspect of the invention, the use of a non-human mammal prepared by the method described in the twelfth or thirteenth aspect of the invention is provided for: (a) Scientific research on retinal diseases; (b) Screening agents for the detection and / or treatment of retinal diseases; and / or (c) Evaluate the effectiveness of methods, formulations and / or drugs for retinal diseases.
[0084] In another preferred embodiment, the non-human mammal prepared by the method described in the twelfth aspect of the present invention is used for: (a) Scientific research on retinal diseases; and / or (d) Screening for retinal pathogenic mutation sites occurring in the second exon of the human RHO gene.
[0085] In another preferred embodiment, the non-human mammal prepared by the method described in the thirteenth aspect of the present invention is used for: (a) Scientific research on retinal diseases; (b) Screening agents for the detection and / or treatment of retinal diseases; and / or (c) Evaluate the effectiveness of methods, formulations and / or drugs for retinal diseases.
[0086] In another preferred embodiment, it is used for: A) Applications in product development involving retinal diseases of human cells; B) Applications as model systems in pharmacological, immunological, microbiological, and medical research; and / or C) Etiological studies involving the production and use of animal experimental disease models, where the applications are for non-disease diagnostic and therapeutic purposes.
[0087] In another preferred embodiment, the retinal lesion refers to the retina RHO Pathogenic gene mutations.
[0088] In another preferred embodiment, the retinal lesion refers to the retina RHO Pathogenic gene mutations that cause structural and / or functional retinal lesions.
[0089] In another preferred embodiment, the drug comprises: a gene therapy drug.
[0090] In another preferred embodiment, the method comprises a gene-editing therapy.
[0091] In the fifteenth aspect of the invention, a cell, tissue, or organ is provided that expresses the humanized RHO recombinant protein of the first aspect of the invention or the RHO recombinant protein mutant of the second aspect of the invention, or the genome of the cell, tissue, or organ contains the polynucleotide of the third or fourth aspect of the invention, or the cell, tissue, or organ is derived from a non-human animal model obtained by the method of the twelfth or thirteenth aspect of the invention. The cells described cannot develop into an animal individual.
[0092] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description
[0093] Figure 1 The diagram shows the breeding protocols for hRHO / hRHO mice and hR135W / hRHO mice.
[0094] Figure 2 This demonstrates the method for determining the results of genotype identification during mouse breeding.
[0095] Figure 3 The image shows a comparison of HE staining in 2-month-old WT, hRHO / hRHO, and hR135W / hRHO mice.
[0096] Figure 4 The image shows a comparison of ERG results for 2-month-old WT, hRHO / hRHO, and hR135W / hRHO mice.
[0097] Figure 5 The results show the results of a comparative study, which show the unmutated sequence of the full-length human RHO CDS inserted at the start codon and followed by the addition of the WPRE element to enhance its expression. Figure 5 A shows a schematic diagram of gene targeting. Figure 5 B shows the retinal characterization results. Figure 5 C and Figure 5 D shows the results of RHO protein expression levels.
[0098] Figure 6 The WB expression level of the RHO protein of the present invention is shown.
[0099] Figure 7 The sequence alignment of the second exon of human RHO and mouse Rho is shown. Detailed Implementation
[0100] Through extensive and in-depth research, the inventors discovered that inserting the full-length, unmutated human RHO CDS sequence at the start codon, followed by the addition of a WPRE element to enhance its expression, resulted in a humanized sequence that exhibited retinopathy and significantly reduced RHO expression even before the introduction of the pathogenic mutation. This indicates that this construction strategy cannot accurately simulate retinopathy caused by RHO mutations. In contrast, by selectively replacing the exon 2 region in the mouse Rho gene to introduce a humanized exon 2 sequence corresponding to the human RHO gene, the animal model showed normal retinal structure and function and normal RHO expression before the introduction of the pathogenic mutation. However, after introducing a hotspot mutation in human retinopathy (e.g., p.R135W), progressive retinal lesions appeared. Based on these findings, this invention was completed.
[0101] This technical approach, when constructing the humanized RHO mutant model, only involves targeted substitution of the exon 2 region in the mouse Rho gene, introducing a humanized exon 2 sequence corresponding to the human RHO gene, while maintaining the remaining gene structure unchanged from the mouse's endogenous sequence. This approach achieves the characteristics of the humanized sequence while minimizing interference with the overall structure and function of the mouse Rho gene, avoiding the introduction of additional unintended mutations that could affect the phenotype due to large-scale sequence substitution. Furthermore, this technical approach constructs a control model containing only the humanized exon 2 without any pathogenic mutations, to distinguish and exclude phenotypic changes that might be caused by the humanized sequence itself. This provides a more precise and controllable in vivo experimental basis for subsequent gene editing technology evaluation and pathogenic mutation functional analysis.
[0102] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.
[0103] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.
[0104] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.
[0105] As used in this article, the term "or a combination thereof" means "or any combination thereof".
[0106] Retinopathy mouse model targeting the RHO gene In existing technologies, various pathogenic point mutations associated with human hereditary retinal diseases have been introduced into the mouse endogenous Rho gene locus via gene knock-in to study the mechanisms of retinal degeneration caused by RHO mutations. However, these mutation models typically introduce only a single base substitution at the corresponding position in the mouse Rho gene, preserving the original mouse gene sequence structure and failing to introduce humanized sequence fragments of the human RHO gene into key coding regions. However, due to differences in base sequence composition, codon usage, and local structure between mouse and human RHO genes, these models cannot accurately reflect the recognition, binding, and editing behavior of gene editing tools in the context of real human RHO sequences. Furthermore, the lack of humanized sequences in this construction method leads to inaccurate evaluation of editing efficiency and editing window. Currently, because existing technologies do not introduce the corresponding coding fragments of the human RHO gene into the mouse genome, parameters such as editing efficiency, editing window range, and base preference obtained from this model are difficult to directly extrapolate to human gene application scenarios, thus limiting the objective evaluation and comparison of gene editing tool performance. On the other hand, this also hinders the optimization and screening of new gene editing technologies at the target sequence level. Existing models containing only mouse point mutations cannot provide a matching testing platform for editing tools designed for human RHO sequences (such as specific target sequence identification or modification strategies), which limits the optimization, iteration and validation of editing technologies at the sequence level.
[0107] Although the RHO gene p.R135W mutation has been widely reported in patients with hereditary retinal degeneration, there is currently no established genetic model of this mutation in mice, and a lack of RHO p.R135W mutant mice available for in vivo studies is also lacking. Due to the absence of corresponding animal models, the pathogenic mechanism, phenotypic characteristics, and feasibility as a gene-editing target of this mutation in vivo are difficult to study systematically, thus limiting the development and validation of gene-editing technologies targeting this mutation.
[0108] The method for constructing animal models of the present invention This invention provides a method for obtaining through gene knock-in technology RHO A method for knocking in the exon 2 gene into a non-human mammalian model, which can be used for research on the mechanisms of retinal diseases and drug screening.
[0109] The humanized exon 2 targeted replacement method provided by this invention can construct a genetic model in mice that is highly consistent with the human RHO target sequence, and can be used to evaluate the editing efficiency, editing window and specificity of gene editing tools in the context of real human sequence.
[0110] The method for constructing a humanized exon 2 mutation model provided by this invention reduces interference with the function of endogenous genes in mice by minimizing the scope of humanization, thereby improving the genetic stability and phenotypic interpretability of the model.
[0111] This invention also provides a method for obtaining a non-human mammalian model with Rho p.R135W mutation knock-in using gene knock-in technology, which can be used for research on retinal disease-related mechanisms and drug screening.
[0112] In summary, this invention provides a paired design strategy that includes a mutation-free humanized control and a p.R135W mutation model, providing a rigorous in vivo control system for gene editing technology development and pathogenic mutation function research, and is suitable for the validation and optimization of various gene editing technologies.
[0113] This invention utilizes a CRISPR / Cas-mediated knock-in method to introduce a pre-designed homologous arm donor sequence into mouse zygotes, achieving precise replacement of exon 2 at the endogenous Rho gene site. The editing results were identified through cross-homologous arm PCR amplification combined with sequencing, and stable humanized exon 2 mice and humanized exon 2+p.R135W mutant mice were obtained through heterozygous breeding. The constructed animal models are stable and reproducible at the genomic level, providing standardized experimental materials for subsequent research.
[0114] Compared with existing techniques that construct point mutation models only in the context of endogenous Rho genes in mice, this invention avoids additional mutations or differences in expression regulation that may be introduced by large-scale humanization by limiting the replacement range to exon 2. At the same time, by setting up a "mutant-free humanized exon 2" control model, the interference of the humanized sequence itself on phenotypic analysis is effectively eliminated, thereby enabling a more accurate assessment of the pathogenic effect of the p.R135W mutation and its characteristics as a gene editing target.
[0115] Sequence alignment of the second exon of human RHO and mouse Rho as follows: Figure 7 As shown.
[0116] The recombinant protein of the present invention The recombinant protein of this invention is made from human... RHO exon 2 replaces mouse endogenous Rho The protein obtained after gene exon 2. The amino acid sequence of the recombinant protein of this invention is shown in SEQ ID NO:1.
[0117] The recombinant protein of the present invention also includes a mutant protein obtained by introducing a pathogenic mutation widely present in RHO (e.g., p.R135W) based on SEQ ID NO:1. In a specific embodiment, the mutant protein has the sequence shown in SEQ ID NO:2.
[0118] The main advantages of this invention include: (a) This invention uses humanized exon 2 targeted replacement to set up a device carrying human-derived exon 2. RHO The wild-type allele humanized mouse (hRHO / hRHO) can serve as a control group for hotspot mutation studies of retinal diseases, thereby enabling a comparison between wild-type (hRHO / hRHO) and mutant (hR135W / hRHO) under the condition of consistent genetic background, effectively eliminating the influence of non-specific factors on the results.
[0119] (b) By accurately introducing the p.R135W point mutation into humanized mice and combining it with homologous arm PCR and sequencing verification, the target mutant allele was accurately identified and its stable inheritance was confirmed.
[0120] (c) The method for constructing humanized mouse models described in this invention significantly improves the reliability and application value of the constructed animal models in simulating human hereditary retinal diseases; in particular, it is of great significance for the study of the mechanism of RHO hotspot mutations and drug screening.
[0121] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.
[0122] Example 1: Construction of humanized mouse models with gene knock-in without RHO pathogenic mutation and humanized mouse models with RHO pathogenic mutation.
[0123] The mouse strain used in this embodiment was C57BL / 6J, and the surrogate mother mice were of the C57BL / 6J strain. The mice were randomly divided into a control group and an experimental group.
[0124] The specific experimental method is as follows: The first step is to identify suitable target sequences within the introns of the target gene and design sgRNAs targeting these sequences. The sgRNA and PAM sequence information is as follows: gRNA-A1: CCCCTGCTAAAACACTAGCC (SEQ ID NO:3), PAM sequence is AGG; gRNA-A2: CTTTGGTTGAGTTCTGGCCG (SEQ ID NO:4), PAM sequence is TGG; gRNA-B1: CCCTGCTAAAACACTAGCCA (SEQ ID NO:5), PAM sequence is GGG; gRNA-B2: TTTGGTTGAGTTCTGGCCGT (SEQ ID NO:6), PAM sequence is GGG.
[0125] The second step is the design of the genome recombination sequence. A donor sequence containing only humanized Exon 2 and without pathogenic mutations was constructed and named the hRHO-donor sequence. Homologous recombination was then used to achieve endogenous humanization in mice. Rho The humanized replacement of Exon 2 gene, and the hRHO-donor sequence of the control mouse are as follows: acacacacacacatacacacacacacccaggttctggatagaagctggggtaccatgccggtgagcttgtctctgtggggtcagacccaggccacatctaccatccaggattcttgttggtag cactcctgttattcagaagtttgttacgtggtccttccccacactgggcttctgaggctgacatatggactaatgtctggagccccctctttacccatcttcttcccctgctaaaacactagcca gggtgtggccctaagccccagctccaggcactccgaggcagtctctcatgagcctaaagctctaaccaaacagaagagcttctgttttggcacacgggttcttcaccccatccctttctcctcgc cagcccaaactcactgcagtcgctaaggcttggatcaagcctcaaaccagaagcttgcatcctagcctgctctctctgaggtgaggttagagctggaggactgacggctactaactgccttacag GTGAAATTGCCCTGTGGTCCTTGG TGGTCCTGGCCATCGAGCGGTACGTGGTGGTGTGTAAGCCCATGAGCAACTTCCGCTTCGGGGAGAACCATGCCAT CATGGGCGTTGCCTTCACCTGGGTCATGGCGCTGGCCTGCGCCGCACCCCCACTCGCCGGCTGGTCCAG gtaatggcactgagtatcgggtctggcaaggtctttgtgggattccctttgaggacacagagccctcggattggttccaggcataatgtaacatggtattgccccccgaaaaccatcctggtgact ttcccaggctaaggtctaaggtaggggagaagagagggactgaatggtccaatcagtcttattccatgtctgagacccataacaaggagaaccctggacattccaacccttcaccttggccgagtc cctaatcctcggctaagccaaggccaaaccacaatcctctttggttgagttctggccgtgggcctctctctctcttcctctctctctctcactcaccttggaccttagccccttggagaggctgaaccttcccaaaatgcatggtgacattgtagccccaggaactgggtcccatccagcctccaggccaccatatctaaatgagacaagagaaggttgggacagtggtttggacacctagacaggcta (SEQ ID NO:7, where the bold italic underlined part is the second exon of the wild-type human RHO gene).
[0126] The third step is the preparation of Cas9 mRNA and the donor vector. Cas9 mRNA is prepared using conventional methods, and the hRHO donor sequence (SEQ ID NO:7) is cloned into the vector to construct the gene recombination donor vector.
[0127] The fourth step is microinjection of fertilized eggs. sgRNA, Cas9 mRNA and the donor vector are mixed and microinjected into fertilized eggs of C57BL / 6J mice, which are then transplanted into surrogate mothers to obtain F0 generation mice.
[0128] F0 generation positive control heterozygous mice were mated with wild-type C57BL / 6J mice to obtain F1 generation heterozygotes; F1 generation heterozygotes were intercrossed, and F2 generation mice were selected to obtain hRHO / hRHO homozygous control mice.
[0129] Example 2: Rho Genotyping of knock-in mice.
[0130] Genomic DNA was extracted and genotypes were identified from the mice obtained in Example 1. The following primers were used to identify the genotype: F2: 5'-CTTGGATCAAGCCTCAAACCAGAAG-3' (SEQ ID NO: 8); R2: 5'-TCTTCTCCCCTACCTTAGACCTTAG-3' (SEQ ID NO:9); Since both the wild-type band (the original mouse Rho gene) and the positive band (the humanized Rho gene) are 425 bp, they cannot be distinguished by electrophoresis. Therefore, the PCR products were sent for sequencing, and the sequencing primers are shown in SEQ ID NO:9.
[0131] The sequencing results are as follows: (1) hRHO / hRHO homozygous: hRHO: GTGAAATTGCCCTGTGGTCCTTGGTGGTCCTGGCCATCGAGCGGTACGTGGTGGTGTGTAAGCCATGAGCAACTTCCGCTTCGGGGAGAACCATGCCATCATGGGCGTTGCCTTCACCTGGGTCATGGCGCTGGCCTGCGCCGCACCCCCACTCGCCGGCTGGTCCAG (SEQ ID NO: 10) (2) hRHO / mRho heterozygotes: hRHO: GTGAAATTGCCCTGTGGTCCTTGGTGGTCCTGGCCATCGAGCGGTACGTGGTGGTGTGTAAGCCATGAGCAACTTCCGCTTCGGGGAGAACCATGCCATCATGGGCGTTGCCTTCACCTGGGTCATGGCGCTGGCCTGCGCCGCACCCCCACTCGCCGGCTGGTCCAG (SEQ ID NO: 10) mRho: GTGAAATCGCCCTGTGGTCCCTGGTGGTCCTGGCCATTGAGCGCTACGTGGTGGTCTGCAAGCCGATGAGCAACTTCCGCTTCGGGGAGAATCACGCTATCATGGGTGTGGTCTTCACCTGGATCATGGCGTTGGCCTGTGCTGCTCCCCCACTCGTTGGCTGGTCCAG (SEQ ID NO: 11) (3) mRho / mRho wild type: mRho: GTGAAATCGCCCTGTGGTCCCTGGTGGTCCTGGCCATTGAGCGCTACGTGGTGGTCTGCAAGCCGATGAGCAACTTCCGCTTCGGGGAGAATCACGCTATCATGGGTGTGGTCTTCACCTGGATCATGGCGTTGGCCTGTGCTGCTCCCCCACTCGTTGGCTGGTCCAG (SEQ ID NO: 11) Sequencing results and corresponding mouse genotype correspondences are shown in [link to data]. Figure 2 Sequencing confirmed the availability of hRHO / hRHO homozygous, hRHO / mRho heterozygous, and mRho / mRho wild-type mice, all with stable and heritable genotypes.
[0132] Example 3: Rho Retinal characterization in knock-in mice.
[0133] Ocular lateral lenticule (ONL) damage in mice was assessed using hematoxylin and eosin (HE) staining. The specific method was as follows: three eyes per group were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and each section was 5 μm thick. Hematoxylin and eosin (HE) staining was performed. ONL thickness was measured three times at a distance of 1000 μm from the optic disc, and the average value was used to assess ONL damage. The HE staining results of 2-month-old WT mice and 2-month-old hRHO / hRHO mice are shown below. Figure 3 As shown: No obvious atrophy was observed in the outer nuclear layer of hRHO / hRHO mice.
[0134] Light-sensitive function in mice was assessed using electroretinography (ERG). The specific method was as follows: Full-field ERG was recorded using the Celeris rodent electrophysiological system (Diagnosys LLC, Lowell, Massachusetts, USA). Mice were dark-acclimated for at least 12 hours prior to testing. Animals were anesthetized by intraperitoneal injection of aphthylamine under dark-red light conditions, and mydriasis was achieved using a mixture of 0.5% tropicamide and 0.5% phenylephrine hydrochloride eye drops (Medoli-P, Santen Pharmaceutical). The corneal surface was protected and electrically coupled using 0.3% hydroxypropyl methylcellulose gel (GenTeal, Alcon). Dark-acclimated ERG responses were assessed by stimulation with white light flashes of increasing intensity (-2.0, -1.5, -1.0, -0.5, 0, 0.5, and 1.0 log cd sm). -2 Induced. Before the electroretinography test, the animals were first subjected to 1.5 log cd sm -2 Adapt to background light for 10 minutes, then apply -1.0, 0, 1, and 2 log cd sm under the same background light. -2Intensity of flashing stimulation. Data were processed using Espion V6 analysis software (Diagnosys LLC, Lowell, Massachusetts, USA). ERG values of 2-month-old WT mice and 2-month-old hRHO / hRHO mice were compared. Figure 4 As shown, no significant impairment of photoreceptor cell function was observed in hRHO / hRHO mice.
[0135] Example 4: Construction of Rho p.R135W mutant knock-in mouse model.
[0136] The first step is to identify suitable target sequences within the introns of the target gene and design sgRNAs targeting these sequences. The sgRNA and PAM sequence information is as follows: gRNA-A1: CCCCTGCTAAAACACTAGCC (SEQ ID NO:3), PAM sequence is AGG; gRNA-A2: GTGGGCTCGCTGCTGGCATC (SEQ ID NO:12), PAM sequence is AGG; gRNA-B1: CCCTGCTAAAACACTAGCCA (SEQ ID NO:5), PAM sequence is GGG; gRNA-B2: TGGGCTCGCTGCTGGCATCA (SEQ ID NO:13), PAM sequence is GGG.
[0137] The second step is the design of the genome recombination sequence. The pathogenic mutation RHO c.403C>T (p.R135W) was introduced into the humanized Exon 2 sequence to construct a donor sequence containing the humanized Exon 2 and the pathogenic mutation, named the hR135W-donor sequence. Humanization of the mouse endogenous Rho gene Exon 2 was achieved through homologous recombination. The hR135W-donor sequence is as follows: acacacacacacatacacacacacacacccaggttctggatagaagctggggtaccatgccggtgagcttgtctctgtggggtcagacccaggccacatctaccatccaggattcttgttggtagcactcctgttattcagaagtttgttacgtggtccttccccacactgggcttctgaggctgacatatggactaatgtctggagccccctctttacccatcttcttcccctgctaaaacactagccagggtgtggccctaagccccagctccaggcactccgaggcagtctctcatgagcctaaagctctaaccaaacagaagagcttctgttttggcacacgggttcttcaccccatccctttctcctcgccagcccaaactcactgcagtcgctaaggcttggatcaagcctcaaaccagaagcttgcatcctagcctgctctctctgaggtgaggttagagctggaggactgacggctactaactgccttacag GTGAAATTGCCCTGTGGTCCTTGG TGGTCCTGGCCATCGAGTGGTACGTGGTGGTGTGTAAGCCCATGAGCAACTTCCGCTTCGGGGAGAACCATGCCAT CATGGGCGTTGCCTTCACCTGGGTCATGGCGCTGGCCTGCGCCGCACCCCCACTCGCCGGCTGGTCCAG gtaatggcactgagtatcgggtctggcaaggtctttgtgggattccctttgaggacacagagccctcggattggttccaggcataatgtaacatggtattgccccccgaaaaccatcctggtgact ttcccaggctaaggtctaaggtaggggagaagagagggactgaatggtccaatcagtcttattccatgtctgagacccataacaaggagaaccctggacattccaacccttcaccttggccgagtc cctaatcctcggctaagccaaggccaaaccacaatcctctttggttgagttctggccgtgggcctctctctctcttcctctctctctctcactcaccttggaccttagccccttggagaggctgaaccttcccaaaatgcatggtgacattgtagccccaggaactgggtcccatccagcctccaggccaccatatctaaatgagacaagagaaggttgggacagtggtttggacacctagacaggcta (SEQ ID NO:14, where the bold italicized underlined characters are human-borne) RHO (Second exon of gene R135W mutation) The third step is the preparation of Cas9 mRNA and the donor vector. Cas9 mRNA is prepared using conventional methods, and the hR135W donor sequence (SEQ ID NO:14) is cloned into the vector to construct the gene recombination donor vector.
[0138] The fourth step is microinjection of fertilized eggs. sgRNA, Cas9 mRNA and the donor vector are mixed and microinjected into fertilized eggs of C57BL / 6J mice, which are then transplanted into surrogate mothers to obtain F0 generation mice.
[0139] F0 generation positive point mutation heterozygous mice were mated with wild-type C57BL / 6J mice to obtain F1 generation heterozygous point mutation mice hR135W / mRho.
[0140] F1 generation heterozygous point mutant mice hR135W / mRho and F2 generation hRHO / hRHO homozygous control mice obtained in Example 1 were mated to obtain hR135W / hRHO mice. Figure 1 ).
[0141] Example 5: Identification of the Rho p.R135W mutant knock-in mouse model.
[0142] Following the method described in Example 2, genomic DNA was extracted and genotypes were identified from the mice obtained in Example 3. The primers shown below were used to identify the genotypes: F2: 5'-GAAGAGCTTCTGTTTTGGCACAC-3' (SEQ ID NO: 15) R2: 5'-TCAAAGGGAATCCCACAAAGACC-3' (SEQ ID NO:16) Since both the wild-type band (hRHO wild-type and mRho wild-type) and the positive band (hR135W) are 399 bp, they cannot be distinguished by electrophoresis. Therefore, the PCR products were sent for sequencing, and the sequencing primers are shown in SEQ ID NO:16.
[0143] The sequencing results are as follows: (1) hR135W / hRHO heterozygote: hR135W: GTGAAATTGCCCTGTGGTCCTTGGTGGTCCTGGCCATCGAGTGGTACGTGGTGGTGTGTAAGCCATGAGCAACTTCCGCTTCGGGGAGAACCATGCCATCATGGGCGTTGCCTTCACCTGGGTCATGGCGCTGGCCTGCGCCGCACCCCCACTCGCCGGCTGGTCCAG (SEQ ID NO: 17) hRHO wild type: GTGAAATTGCCCTGTGGTCCTTGGTGGTCCTGGCCATCGAGCGGTACGTGGTGGTGTGTAAGCCATGAGCAACTTCCGCTTCGGGGAGAACCATGCCATCATGGGCGTTGCCTTCACCTGGGTCATGGCGCTGGCCTGCGCCGCACCCCCACTCGCCGGCTGGTCCAG (SEQ ID NO: 10) (2) hRHO / mRho heterozygotes: hRHO wild type: GTGAAATTGCCCTGTGGTCCTTGGTGGTCCTGGCCATCGAGCGGTACGTGGTGGTGTGTAAGCCATGAGCAACTTCCGCTTCGGGGAGAACCATGCCATCATGGGCGTTGCCTTCACCTGGGTCATGGCGCTGGCCTGCGCCGCACCCCCACTCGCCGGCTGGTCCAG (SEQ ID NO: 10) mRho wild type: GTGAAATCGCCCTGTGGTCCCTGGTGGTCCTGGCCATTGAGCGCTACGTGGTGGTCTGCAAGCCGATGAGCAACTTCCGCTTCGGGGAGAATCACGCTATCATGGGTGTGGTCTTCACCTGGATCATGGCGTTGGCCTGTGCTGCTCCCCCACTCGTTGGCTGGTCCAG (SEQ ID NO: 11) Sequencing results and corresponding mouse genotype correspondences are shown in [link to data]. Figure 2 Sequencing confirmed the successful acquisition of hR135W / hRHO mutant mice, with the mutation site showing stable inheritance.
[0144] Example 6: Retinal characterization of a Rho p.R135W mutant knock-in mouse model.
[0145] Following the method described in Example 3, the hR135W / hRHO mutant mice were subjected to HE staining and ERG detection.
[0146] HE staining results of 2-month-old WT mice and 2-month-old hR135W / hRHO mice are as follows: Figure 3 As shown, the outer nuclear layer of hR135W / hRHO mice is significantly atrophied.
[0147] ERG of 2-month-old WT mice and 2-month-old hR135W / hRHO mice as follows Figure 4 As shown, the photoreceptor cell function of hR135W / hRHO mice is significantly impaired.
[0148] discuss This invention first provides a method for constructing a humanized RHO mutant animal model by targeted humanization replacement of exon 2 (exon 2) of the mouse Rho gene. This method uses the mouse endogenous Rho gene as the editing target site, introducing a humanized sequence fragment identical to the human RHO gene only in the exon 2 region, while maintaining the remaining gene structure and regulatory regions unchanged from the mouse endogenous sequence. This approach minimizes interference with the overall structure and function of the mouse Rho gene while introducing humanized target sequence characteristics. The amino acid sequence of the recombinant protein obtained after targeted humanization replacement of exon 2 (exon 2) of the mouse Rho gene is shown below: MNGTEGPNFYVPFSNVTGVVRSPFEQPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLAGWS RYIPEGMQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIVIFFCYGQLVFTVKEAAAQQQESATTQKAEKEVTRMVIIMVIFFLICWLPYASVAFYIFTHQGSNFGPIFMTLPAFFAKSSSIYNPVIYIMLNKQFRNCMLTTLCCGKNPLGDDDASATASKTETSQVAPA* (SEQ ID NO:1).
[0149] Building upon this, the present invention further introduces the pathogenic mutation p.R135W, widely present in the RHO gene, into the humanized exon 2 sequence to construct a mouse model carrying both humanized exon 2 and the p.R135W mutation. Simultaneously, to eliminate the potential impact of the humanized sequence itself on the retinal structure or function of the mice, the present invention also constructs a control mouse model containing only humanized exon 2 without any pathogenic mutation. This control model allows for the differentiation between the background effect of the humanized sequence and the phenotypic changes caused by the p.R135W mutation, providing a reliable control basis for mutation function studies and gene editing effect evaluation. The amino acid sequence of the p.R135W mutated protein is shown below: MNGTEGPNFYVPFSNVTGVVRSPFEQPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIEWYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLAGWS RYIPEGMQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIVIFFCYGQLVFTVKEAAAQQQESATTQKAEKEVTRMVIIMVIFFLICWLPYASVAFYIFTHQGSNFGPIFMTLPAFFAKSSSIYNPVIYIMLNKQFRNCMLTTLCCGKNPLGDDDASATASKTETSQVAPA* (SEQ ID NO:2).
[0150] It should be particularly noted that the animal model construction scheme of this invention was obtained through a very large number of experiments and explorations. In the preliminary experiments, we attempted to insert the full-length unmutated human RHO CDS sequence at the start codon and then add a WPRE element to enhance its expression. Figure 5 A). Experiments showed that homozygotes without pathogenic mutations exhibited significant shortening of the outer nuclear layer after 3 months. Figure 5 B), Western blot analysis at 3 weeks showed that the expression level of RHO protein in homozygotes was only 0.1 times that of wild-type. C and D). In this invention, such as As shown, after simply replacing exon 2 with humanized material, the expression level of RHO protein was not significantly different from that in wild-type mice, and the retina showed almost no deterioration. Furthermore, by introducing pathogenic mutations (such as RHO p.R135W) into humanized mice, pathogenic mutations in humans could be efficiently simulated. This was quite unexpected.
[0151] In summary, this invention, based on existing RHO mutant animal models, is the first to achieve a technical solution that realistically simulates the human RHO target sequence background in vivo by replacing only exon 2 with humanized material and simultaneously constructing a mutation-free control model. This solves the problem of the lack of suitable RHO p.R135W animal models for gene editing development in existing technologies. This technical solution provides a new experimental platform and technical approach for the safety assessment and application development of gene editing tools in hereditary retinal diseases.
[0152] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.
Claims
1. A humanized RHO recombinant protein, characterized in that, The humanized RHO recombinant protein is a chimeric protein obtained by replacing the amino acid region encoded by the second exon in the Rho protein of non-human mammals with the amino acid region encoded by the second exon in the human RHO protein.
2. The RHO recombinant protein as described in claim 1, characterized in that, The recombinant protein contains the sequence shown in SEQ ID NO:
1.
3. A mutant of the RHO recombinant protein, characterized in that, The mutant is a recombinant RHO protein mutant having one or more site mutations selected from the group consisting of: R135W, R135L, P171L, A164V, L125R; The amino acid numbering of the mutation is based on the human RHO protein, whose Genbank accession number is NP_000530.
1.
4. A polynucleotide, characterized in that, The polynucleotide encodes the RHO recombinant protein of claim 1 or the RHO recombinant protein mutant of claim 3.
5. A carrier, characterized in that, The vector comprises the polynucleotide of claim 4.
6. The use of the polynucleotide of claim 4 or the vector of claim 5, characterized in that, Used to prepare non-human animal models.
7. A method for preparing a non-human animal model, characterized in that, Including the following steps: (S1) Providing modified non-human animal pluripotent stem cells expressing the RHO recombinant protein of claim 1 or the RHO recombinant protein mutant of claim 3; and (S2) Use the modified non-human animal pluripotent stem cells described in (S1) to generate non-human animal models.
8. The use of the non-human mammal prepared by the method of claim 7, characterized in that, Used for: (a) Scientific research on retinal diseases; (b) Screening agents for the detection and / or treatment of retinal diseases; and / or (c) Evaluate the effectiveness of methods, formulations and / or drugs for retinal diseases.
9. The use as described in claim 8, characterized in that, The drug includes: gene therapy drugs.
10. A cell, tissue, or organ, characterized in that, The cells, tissues, or organs express the humanized RHO recombinant protein of claim 1 or the RHO recombinant protein mutant of claim 3; or, the genome of the cells, tissues, or organs contains the polynucleotide of claim 4; or, the cells, tissues, or organs are derived from a non-human animal model obtained by the method of claim 7. The cells described cannot develop into an animal individual.