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CN-122012613-A - Construction method and application of transgenic mouse animal model for expressing human ATP7B and high-frequency mutation type thereof

CN122012613ACN 122012613 ACN122012613 ACN 122012613ACN-122012613-A

Abstract

The invention discloses a construction method and application of an animal model of a transgenic mouse expressing human ATP7B and a high-frequency mutant type thereof, belonging to the technical field of animal model construction. The invention provides a construction method of a humanized ATP7B high-frequency mutant transgenic mouse model. Firstly, modifying BAC plasmid, knocking out a non-target gene, and accurately introducing p.R778L, p.P992L and p.T935M point mutations special for eastern Asian population by using CRISPR/Cas9 technology to obtain mutant BAC DNA. Subsequently, it was microinjected into mouse fertilized eggs, and the F0-generation mice that gave positive transgenes were bred. Finally, the target strain expressing only the human mutant ATP7B is obtained by crossing and screening the mice with the Atp B gene knockout through multiple generations, wherein the endogenous Atp B is completely deleted. The model can accurately simulate the genetic background and pathological phenotype of east Asia WD patients, can be used for screening targeted drugs aiming at human ATP7B mutation, and can evaluate the improvement effect of the drugs on liver and nerve injury.

Inventors

  • WU ZHIYING
  • LIU CHONG
  • DONG YI
  • ZHENG ZIWEI

Assignees

  • 浙江大学

Dates

Publication Date
20260512
Application Date
20251230

Claims (10)

  1. 1. The construction method of the transgenic mouse animal model for expressing the human ATP7B and the high frequency mutant thereof is characterized by comprising the following steps: S1, knocking out three additional genes of UTP14C, ALG and NEK5 of BAC plasmids, transforming the ATP7B-BAC plasmids into escherichia coli to obtain BAC strains, then carrying out recombination operation in the strains by utilizing pRedET and pFlpe plasmids to introduce 3 XMyc tags to obtain wild type ATP7B-BAC DNA, respectively carrying out p.R778L, p.P992L and p.T935M base modification on the wild type ATP7B-BAC DNA by adopting CRISPR/Cas9 combined single-strand DNA mediated base editing technology, obtaining 4 ATP7B-BAC DNAs after screening, marker excision and sequencing verification, then carrying out dialysis on the obtained mutant ATP7B-BAC DNA by adopting NotI enzyme digestion, and obtaining purified mutant ATP7B-BAC DNA by adopting a filter; S2, introducing purified mutant ATP7B-BAC DNA into fertilized eggs of donor mice through microinjection, then transplanting the fertilized eggs into a surrogate mother mouse to enable the fertilized eggs to develop and produce F0 mice, and screening out transgenic positive F0 mice; S3, hybridizing the obtained transgenic positive F0 generation mice with Atp B -/- mice to obtain F1 generation mice, backcrossing the F1 generation mice with Atp B -/- mice again, continuously hybridizing for 2 to 3 generations, carrying out genotype identification on the sub generation mice after each generation of hybridization, and screening out mice with genotype being endogenous Atp B homozygous knockout and integrated with human ATP7B mutant BAC, namely, expressing the human ATP7B high-frequency mutant transgenic mice.
  2. 2. The method according to claim 1, wherein p.r778l is located at exon 8 of ATP7B gene in S1, and the base mutation c.233g > T results in the change of amino acid from arginine to leucine; p.992l is located at exon 13, the base mutation c.2975c > T, the amino acid is changed from proline to leucine, the second largest high frequency mutation is the eastern population; T935M is located in exon 12, the base mutation c.2804C > T, the amino acid is changed from threonine to methionine.
  3. 3. The construction method according to claim 1, wherein the single-stranded DNA sequence edited by CRISPR/Cas9 base in S1 is designed for each mutation site, ensuring that only the target base is replaced without other random mutation, and sequencing verification adopts Sanger sequencing, and covers the full-length coding region of the ATP7B gene and 200bp upstream and downstream of the mutation site.
  4. 4. The method according to claim 1, wherein the 4 kinds of ATP7B-BAC DNAs in S1 are respectively ATP7B-BAC DNA not subjected to base modification, ATP7B-BAC DNA subjected to base modification of p.R778L, ATP7B-BAC DNA subjected to base modification of p.P992L, and ATP7B-BAC DNA subjected to base modification of p.T935M.
  5. 5. The method according to claim 1, wherein the concentration of the purified mutant ATP7B-BAC DNA in S1 is not less than 50 ng/. Mu.L.
  6. 6. The method of claim 1, wherein the donor mice in S2 are SPF grade Fvb mice.
  7. 7. The method of claim 1, wherein the microinjection in S2 is a male prokaryote injecting a DNA solution into a fertilized egg.
  8. 8. The method according to claim 1, wherein the Atp b -/- mouse in S3 is an endogenous Atp b knockout mouse.
  9. 9. Use of a transgenic mouse animal model obtained by the construction method according to any one of claims 1-8 for studying the pathogenesis of hepatolenticular degeneration (WD), characterized by being used for studying the influence of high frequency mutations p.r778l, p.p992l, p.t935m of WD on copper metabolism in the eastern asian population, and the occurrence mechanism of the cone external system symptoms of WD neurological symptoms.
  10. 10. Use of the transgenic mouse animal model obtained by the construction method according to any one of claims 1 to 8 in WD therapeutic drug screening and evaluation, characterized in that it is used for evaluating the effect of a targeted drug against high frequency mutations of human ATP7B on improving liver injury, neurological function.

Description

Construction method and application of transgenic mouse animal model for expressing human ATP7B and high-frequency mutation type thereof Technical Field The invention belongs to the technical field of animal model construction, and particularly relates to a construction method and application of a transgenic mouse animal model for expressing human ATP7B and high-frequency mutation thereof. Background Hepatolenticular degeneration (WD) is an autosomal recessive disorder of copper metabolism caused by a mutation in the ATP7B gene resulting in a defect in the function of the encoded copper transport P-type ATPase. Patients clinically manifest liver cirrhosis, neuropsychiatric symptoms and the like, and serious patients can endanger life. The existing animal models for studying WD mainly include the following classes, but all have significant limitations: Thus, there is a long-felt lack in the art of an animal model of WD that is capable of satisfying the following conditions simultaneously: (1) Genetic background matching, namely carrying high-frequency pathogenic mutation of east Asia people accurately; (2) The pathological phenotype is complete, and the pathological characteristics of a plurality of organs such as the liver, the nervous system and the like of WD can be reproduced; (3) Has strong practicability, is based on a mouse system with rapid propagation and low cost, and is convenient for large-scale research. This gap severely restricts the development of pathogenesis studies (particularly the neurosymptomatic mechanisms) and targeted therapeutic drugs for eastern asia WD patients. Disclosure of Invention In view of the above, the present invention aims to provide a method for constructing a transgenic mouse animal model expressing human ATP7B and its high frequency mutation, and its application. In order to achieve the above object, the present invention provides the following technical solutions: the invention provides a construction method of a transgenic mouse animal model for expressing human ATP7B and a high-frequency mutant thereof, which comprises the following steps: S1, knocking out three additional genes of UTP14C, ALG and NEK5 of BAC plasmids, transforming the ATP7B-BAC plasmids into escherichia coli to obtain BAC strains, then carrying out recombination operation in the strains by utilizing pRedET and pFlpe plasmids to introduce 3 XMyc tags to obtain wild type ATP7B-BAC DNA, respectively carrying out p.R778L, p.P992L and p.T935M base modification on the wild type ATP7B-BAC DNA by adopting CRISPR/Cas9 combined single-strand DNA mediated base editing technology, obtaining 4 mutant ATP7B-BAC DNAs after screening, marker excision and sequencing verification, then carrying out enzyme digestion on the obtained mutant ATP7B-BAC DNA by adopting NotI, and dialyzing by a filter to obtain purified mutant ATP7B-BAC DNA; S2, introducing purified mutant ATP7B-BAC DNA into fertilized eggs of donor mice through microinjection, then transplanting the fertilized eggs into a surrogate mother mouse to enable the fertilized eggs to develop and produce F0 mice, and screening out transgenic positive F0 mice; S3, hybridizing the obtained transgenic positive F0 generation mice with Atp B -/- mice to obtain F1 generation mice, backcrossing the F1 generation mice with Atp B -/- mice again, continuously hybridizing for 2 to 3 generations, carrying out genotype identification on the sub generation mice after each generation of hybridization, and screening out mice with genotype being endogenous Atp B homozygous knockout and integrated with human ATP7B mutant BAC, namely, expressing the human ATP7B high-frequency mutant transgenic mice. Further, p.R778L is located at the 8 th exon of the ATP7B gene in S1, the base mutation c.2333G > T causes the amino acid to be changed from arginine to leucine, p.P992L is located at the 13 th exon, the base mutation c.2975C > T causes the amino acid to be changed from proline to leucine, the second highest frequency mutation is carried out on eastern people, p.T935M is located at the 12 th exon, the base mutation c.2804C > T causes the amino acid to be changed from threonine to methionine. Furthermore, the single-stranded DNA sequence edited by CRISPR/Cas9 base in S1 is designed for each mutation site, so that only the target base is replaced without other random mutation, and the sequence verification adopts Sanger sequencing to cover the full-length coding region of the ATP7B gene and 200bp upstream and downstream of the mutation site. Further, the 4 kinds of ATP7B-BAC DNA in S1 are respectively ATP7B-BAC DNA which is not subjected to base modification, ATP7B-BAC DNA which is subjected to base modification of p.R778L, ATP7B-BAC DNA which is subjected to base modification of p.P992L and ATP7B-BAC DNA which is subjected to base modification of p.T935M. Preferably, the concentration of the purified mutant ATP7B-BAC DNA in S1 is not less than 50 ng/. Mu.L. Preferably, the donor mice i