CN117778401A

CN117778401A - Construction method and application of donor segment of targeting hTUBB8 gene and ovum maturation disorder animal model

Info

Publication number: CN117778401A
Application number: CN202311821641.9A
Authority: CN
Inventors: 张天镭; 郑伟; 谷峰; 李卓; 彭梓妍; 林戈
Original assignee: Guang Xiu Gao Xin Life Science Co ltd Hunan; Reproductive and Genetic Hospital of CITIC Xiangya Co Ltd
Current assignee: Guang Xiu Gao Xin Life Science Co ltd Hunan; Reproductive and Genetic Hospital of CITIC Xiangya Co Ltd
Priority date: 2023-12-27
Filing date: 2023-12-27
Publication date: 2024-03-29

Abstract

The application provides a donor segment of a targeted mutation hTUBB8 gene, a construction method of an ovum maturation disorder animal model and application thereof. The donor fragment comprises a ZP3promoter region, a Kozak sequence, a mutated hTUBB8 gene fragment, a tag sequence, a WPRE sequence and a terminator sequence from the 5 'end to the 3' end, wherein the mutated hTUBB8 gene fragment has the following mutation compared with a wild type hTUBB8 gene: c.785G > A, the cDNA sequence of the wild hTUBB8 gene is shown in SEQ ID NO: 3. The animal model constructed by the nucleic acid product can provide help for deeply analyzing the pathogenicity of TUBB8 mutation and exploring a treatment strategy, and provides a new reference for diagnosing and treating patients with the gene defects clinically.

Description

Construction method and application of donor segment of targeting hTUBB8 gene and ovum maturation disorder animal model

Technical Field

The application relates to the technical field of animal models, in particular to a construction method and application of a donor segment of a targeting hTUBB8 gene, an ovum cell maturation disorder animal model.

Background

In recent years, infertility has become one of the major diseases affecting physiological and psychological health of humans, and about 15% of couples worldwide have suffered from it, with female infertility being the major factor. Oocyte maturation is subjected to a series of complex physiological changes such as meiosis recovery, reproduction bubble disappearance, polar body discharge and the like, and molecular regulation and the like, so that the potential of fertilization and development into new individuals can be obtained. When the oocyte is abnormal in the maturation process of the egg cell due to various reasons, the oocyte cannot be matured and the fertilization process cannot be completed, thereby resulting in infertility. Clinically, it is not uncommon to have recurrent complete oocyte maturation disorders. The main type is primary infertility caused by repeated maturation retardation of most oocytes in different IVF/ICSI cycles, which is one of the reasons why a few women still fail to receive assisted reproductive technology for multiple times. With the widespread use of second generation sequencing technology, a number of pathogenic genes have been discovered that lead to the developmental maturation disorder of human eggs. However, at present, there is no effective treatment method for ovum maturation disorder in clinic, and the pathogenesis of many female infertility patients is not clear.

Therefore, the search for new pathogenic genes and new animal models is of great importance for clinical diagnosis and treatment of diseases associated with female infertility caused by ovum maturation disorders.

Disclosure of Invention

The present application found that the TUBB8 (c.G785A) mutation has a dominant negative effect, which can disrupt microtubule function and assembly of the meiotic spindle of the oocyte, resulting in female infertility. Based on the above, the application designs a simple and effective mode, and uses the Crispr-Cas system to quickly knock in the hTUBB8 gene to construct an animal model of the disease. According to the method, the ZP3promoter is used for driving the expression of TUBB8 (c.G785A) genes, the Crispr-Cas system can accurately cut DNA to cause double strand break under the guidance of gRNA, and the hTUBB8 (c.G785A) genes can be specifically knocked in for mutation, so that an animal model for female infertility is constructed.

The specific scheme of the application is as follows:

a first aspect of the present application provides a donor fragment targeting the hTUBB8 gene, said donor fragment comprising, in order from the 5 'end to the 3' end, a ZP3promoter region, a Kozak sequence, a mutated hTUBB8 gene fragment, a tag sequence, a WPRE sequence and a terminator sequence, wherein the mutated hTUBB8 gene fragment has the following mutations compared to the wild-type hTUBB8 gene: c.785G > A, the cDNA sequence of the wild hTUBB8 gene is shown in SEQ ID NO: 3.

In one embodiment, the nucleotide sequence of the donor fragment is shown in SEQ ID NO. 2.

An hTUBB8 gene editing system comprising the above-described donor fragment targeting the hTUBB8 gene;

optionally, the editing system further comprises one or more of Cas9 nuclease, cas9mRNA, and gRNA.

A recombinant cell comprising a donor fragment targeted to the hTUBB8 gene as described above or an hTUBB8 gene editing system as described above.

The application also provides a construction method of the ovum maturation disorder animal model, which comprises the following steps:

an ovum maturation disorder animal model was constructed using the hTUBB8 gene editing system described above.

In one embodiment, the construction method comprises the steps of:

transferring the hTUBB8 gene editing system into fertilized eggs of target animals;

transplanting the fertilized egg transferred into the hTUBB8 gene editing system into female animals and producing F0 generation; and

mating the F0 generation with a wild type to obtain an F1 generation heterozygote, selfing the F1 generation heterozygote, and screening out a homozygous F2 generation serving as an ovum maturation disorder animal model.

In one embodiment, the method of transferring comprises microinjection.

In one embodiment, the construction method further comprises the steps of:

the primer pair with the nucleotide sequence shown as SEQ ID NO. 4-7 is used for identifying the hTUBB8 genotype of the target animal.

In one embodiment, the method of identifying comprises one or more of PCR and Sanger sequencing.

The expression of TUBB8 (c.G785A) gene is driven by the ZP3promoter, so that the expression characteristics of TUBB8 mutant patients are simulated. The application provides a construction method of a hTUBB8 (c.G785A) gene knock-in mouse model, which utilizes a Crispr-Cas system to accurately cut DNA under the guidance of gRNA to cause double-strand break, can specifically knock in the hTUBB8 (c.G785A) gene mutation, and accurately simulates the mutation site of a TUBB8 mutation patient. Therefore, an effective mouse model capable of simulating TUBB8 expression characteristics and mutation sites carried by patients is constructed, help is provided for in-depth analysis of pathogenicity of the mutation and exploration of treatment strategies, and a new reference is provided for diagnosis and treatment of patients with the gene defects clinically.

The application also provides a method for screening or identifying a drug for treating an egg maturation disorder, which uses the egg maturation disorder animal model prepared by the method for constructing an egg maturation disorder animal model.

Drawings

FIG. 1 is a schematic diagram of the genotype of a Mouse knocked-in hTUBB8 (c.G785A) gene in example 1 of the present application, wherein Mouse ZP3promoter represents the Mouse ZP3promoter region, kozak-Human TUBB8 (c.G785A) CDS-Myc tag represents the region of Kozak sequence, hTUBB8 (c.G785A) CDS sequence and Myc tag sequence in this order from the 5 'end to the 3' end, WPRE is a cis-acting RNA element, is a post-transcriptional regulatory sequence, BGH pA represents the transcription termination signal region, and arm represents the homology arm region;

FIG. 2 is a graph showing the identification result of F2 mice in example 1 of the present application;

FIG. 3 is a graph showing the results of HE staining of hTUBB8 (c.G785A) knock-in mice.

Detailed Description

The detailed description of the embodiments of the present application will be presented in order to make the above objects, features and advantages of the present application more obvious and understandable. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. This application is, however, susceptible of embodiment in many other forms than those described herein and similar modifications can be made by those skilled in the art without departing from the spirit of the application, and therefore the application is not to be limited to the specific embodiments disclosed below.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, and the like used in the present application are commercially available or may be prepared by existing methods.

Cas9mRNA can transiently express Cas9 protease, and the Cas9 protease is characterized by being capable of autonomously combining and cutting target genes, and inactivating two structural domains RuvC-and HNH-of the Cas9 in a point mutation mode, so that the formed Cas9 can only combine with target genes under the mediation of sgRNA, and has no function of cutting DNA.

The regular clustered interval short palindromic repeat (CRISPR/Cas 9) technology is established based on immune system modification in bacteria and archaea, consists of an R-S structure formed by a plurality of regular short palindromic sequences and non-repeated interval sequences and a gene operator for encoding Cas9 nuclease, and is characterized in that target DNA sequence identification is carried out and DNA double strand break is caused by guiding RNA (gRNA) -mediated endonuclease Cas9 protein, damaged DNA is restored in a homologous recombination or non-homologous end connection mode, and thus, multiple modifications such as site-directed knockout, knock-in and gene correction of genes are realized on target sites. The CRISPR/Cas9 technology has been rapidly developed in recent years due to the characteristics of high specificity, simple molecular construction, short flow and the like. Compared with Zinc Finger Nuclease (ZFN) technology and transcription-like effector nuclease (TALEN) technology, the method has more and more wide application due to the advantages of specificity, high efficiency, simplicity in design and the like of the target editing target genes, and has strong genome editing activity in bacteria, mammalian cells, zebra fish, mice, rats and the like.

The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from "and/or", "or/and", "and/or", it should be understood that, in this application, the technical solutions certainly include technical solutions that all use "logical and" connection, and also certainly include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical scheme of "logical or" connection), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical scheme of "logical and" connection).

An embodiment of the present application provides a donor fragment targeting the hTUBB8 gene, the donor fragment comprising, in order from the 5 'end to the 3' end, a ZP3promoter region, a Kozak sequence, a mutated hTUBB8 gene fragment, a tag sequence, a WPRE sequence, and a terminator sequence, wherein the mutated hTUBB8 gene fragment has the following mutation c.785g > a compared to the wild-type hTUBB8 gene, and the cDNA sequence of the wild-type hTUBB8 gene is as set forth in SEQ ID NO: 3.

Specifically, the sequence set forth in SEQ ID NO:3 is: 5'-ATGAGGGAGATCGTGCTCACGCAGATCGGGCAGTGCGGGAATCAGATCGGCGCCAAGTTCTGGGAGGTGATCTCTGATGAACATGCCATCGACTCCGCTGGCACCTACCACGGGGACAGCCACCTGCAGCTGGAGCGCATCAACGTGTACTACAACGAGGCCAGCGGTGGCAGGTACGTGCCCCGCGCTGTGCTCGTGGATCTGGAGCCGGGCACCATGGACTCTGTGCGCTCGGGGCCCTTCGGGCAGGTCTTCAGGCCAGACAACTTCATCTTCGGTCAGTGTGGGGCCGGAAACAACTGGGCCAAGGGACACTACACCGAAGGCGCGGAGCTGATGGAGTCAGTGATGGACGTTGTCAGAAAGGAGGCTGAGAGCTGTGACTGCCTGCAGGGTTTCCAGCTGACCCACTCCCTGGGTGGGGGGACTGGGTCTGGGATGGGTACCCTTCTGCTCAGTAAGATCCGGGAGGAGTACCCAGACAGGATCATAAACACATTCAGCATCCTGCCCTCGCCCAAGGTGTCGGACACCGTGGTGGAGCCCTACAACGCCACCCTCTCAGTCCACCAGCTCATAGAAAACGCAGATGAGACCTTTTGCATAGATAACGAAGCTCTGTATGACATATGTTCCAAGACCCTAAAACTGCCCACACCCACCTATGGTGACCTGAACCACCTGGTGTCTGCTACCATGAGTGGGGTCACCACGTGCCTGCGCTTCCCGGGCCAGCTGAATGCTGACCTGCGGAAGCTGGCCGTGAACATGGTCCCGTTTCCCCAGCTGCATTTCTTCATGCCCGGCTTTGCCCCACTGACCAGCCGGGGCAGCCAGCAGTACCGGGCCTTGACTGTGGCTGAGCTTACCCAGCAGATGTTTGATGCTAAGAACATGATGGCTGCCTGTGACCCCCGTCACGGCCGCTACCTAACGGCGGCTGCCATTTTCAGGGGTCGCATGCCCATGAGGGAGGTGGATGAACAAATGTTCAACATTCAAGATAAGAACAGCAGTTACTTTGCTGACTGGCTCCCCAACAACGTAAAAACAGCCGTCTGTGACATCCCACCCCGGGGGCTAAAAATGTCAGCCACCTTCATTGGGAATAATACGGCCATCCAGGAACTCTTCAAGCGTGTCTCAGAGCAGTTTACAGCAATGTTCAGGCGCAAGGCCTTCCTCCACTGGTACACGGGCGAGGGCATGGATGAGATGGAATTCACCGAGGCCGAGAGCAACATGAACGACCTGGTGTCTGAATATCAGCAATATCAGGATGCCACGGCCGAGGAGGAGGAGGATGAGGAGTATGCCGAGGAGGAGGTGGCCTAG-3'.

In some of these embodiments, the nucleotide sequence of the donor fragment is shown in SEQ ID NO. 2.

Specifically, the nucleotide sequence shown in SEQ ID NO. 2 is:

5'-CTTGTACCCTTTGGTCTCTGCTGCTAGTCACCCTCTCCTAAACCCTCGTCCTGGGACCCCTCTGCATTTGGCGGTGATGCAGCAGCAGCTACAGCGCTCAGGTAAGGTTTAGAAATAGGCCAGGTTAGCTGATCACACTGGAAAGTTCTGAGATTTCCAGGAGCAATAGTTATCACTGCCCCTTTGTTTCTTAGGTGTCACTAGAAAAACAGGTGAAGTGAACTGACCTGTGCAGTCTCAGGGATTATTACTACTGCAAGGACAGTCGGAGCCAGCCATGCTCTAGCTCTGTTTTTCACTTCTTATAAGCCTGAGAATTTTTGCTGAGATGTGAACATGTCAGCCTTGGTGGGCTTTCTTACGTTGCATTTTCTTACGTTGCATTTTGCACTTTAGGAAGCCTTTGGTTAAATCCTCTGGTAGACTTCCCATCCCCCAAAGTAACAAACCATTCAAGCAAAGTAGGTCAGAAAATATTGTCAATACTGAGAACAGGACTTGGGAGTGAGTATCCAAGGGTGGTCTTAGTAGACTTGTGGGGTTCTGTGGGTGAGGTGTAGAGTAGAGCTACTTTGCTGCACTGACACTCTTCTCTCCATAGTTCTGCATCCTCCAGGCTCTAGTTCCCAGGCAGCAGCTATCAGCGTTCAGACTCCTCAGAATGTACCCAGCCGGTCGGGCATGCCCCACATGCACTCCCAGCTGGAGCATCGTACCAGCCAAAGGAGCAGCTCCCCTGTGGGCCTTGCCAAATGGTTTGGCTCAGATGTGCTACAGCAGCCTCTGCCCTCCATGCCCACCAAAGTCATCAGTGTAGATGAACTGGAATATAGACAGTGAAGAGGGCAGGCTGGCTCACCCATACCTGGACCTGTGGTGACACCCTGGTCATGACTCTCATTCCCTCTTTGTAATGGGCTTTTACATTGGAGCACACTATGTGAAGATGTTTAGGGGATCCACATACCTACCATGATCTACATTATGACAGAAGGCTGTTAAATCGAATGAACCTACATGGTTCAAATACAAGGGATACAAGATTGTCAGTCCTGGAAGTCTTTCTTTTATAAAATATGTGAATGAAGTGTTGGTGTCTTCTAGAGGTGACACCTAAGGGTTCTGAAAAAATAAAATGTATAGACCCTTATGTACAGACCTGTGTATAAACTTTTGTACATACAAATAGGGTAGCTTTTTTTGAACTTATACATACAGCTGTACATAAAGTAACTATCAGTTAGGCTTGTGTCAACTGTTTGGATTTTTTTCACTTGAATATTTGGGACTTTTTCTTTTGGTTTATTAAAAGTTACATATGCCACGTGTGTGAACGATATGGCTGGTACTGTGTTTATTTCTTCCATGAACTAAGACAGTCTAAATGAGTTCCTTTCACGTTTTAATTTTACCTTAGGACTTCTGGAATTTCTTCTGCACATAAAGTTCTGATAGCATTAGTTTAAGCTGGACTAACCCTGAAAGTAGCTTGTGGCAAGTATCAAGGAATCAATATTATACTCTACAAAATCAAAGTTTACAGAGAAGTCATATAGTAATTTTTCTGAAATTTACTGGCACAATGTTAATCCAGCCTGACTCCAACTAATTAATGGTCACATTAATTTAAGTCTTTCCCTTGCCTCTGCTGCATTAGTTTCTCTCAAAATTGTTAACTTACAACTTGAAGTCTGGTATTATAAATTGAATGTAAAGCATTCTGAAAGATACTATACTGATTGCAGGTTTTTCAGTCAGGTTCAAGCTAATTTGACCAGTCATTGGATTAATTATGGATCTGGGGCCATAAATGCTATTTTAATTCCACTATAGAGATTAAAATAAGCCATTCTCCATTTCATAATATTCTATTGGACTTTGACTGCAGGGGCCTCCAAGTCTTGACAGTAGATTATAATCCTTCAGCTGCCCACTCTACTGGAGGAGGACAAACTGGTCACTTTTCAGCAAAACCTGGCTGTGGATCAGGGCAGTCTGGTACTTCCAAGCTCATTAGATGCCATCATGCTCTCACTGCCTCCTCAGCTTCAAGAGGAATCTGGAAAAAGCAGTCCCACTGGTCAGGAAAGGAACACTAGTGCACGGCGCGCCAATTCCTTTTAGCCCCGGTGGGGCAGGTGATTAGGAGCTGTGAAGCCTTTGTTCTTATTGGCAAAAGGGTCCCATATTCTTTCCCTTATACTTGTTGCCATATTAAATGGGTAATGTTTATAAAAGGTTTCCCCAGTGTCTGGCCCGCTCTAATGTGCTTAGCTATTGCTATGACTGCCCCCGTCTGACCTCCAAGACAAGAGCCAGATCTCATTCAATAGTTGTACGTGGCATATGATGTTCAGAAAATGTTTGTTGATGGGCTGAGTGAAATTGTGGCTATGAAGTCATAGAATGAAACAGAGGGGTCTCAGGAAGTAGTCAATGAGTTGGCTCAGCAGGTAAAGGTGACTGCTGCCAAGTCTGATGACCTAAGTTCAATTCCTGGGACCCAAATGTCAGAAGGCAAAACACTCTTGCAAGTTGCCCTGTGACTGTCACTTGTGCATGCCTGCACACACATAAATAAATAAATGCAATATTAAGAATTTTTTTTTTTTTAAAGAAAGGGCTCTGGAGGTTTTCAAAGGAAATTGTTATTACACAGAGAGAACACACACACACACACACACACACACACACACACAACTGAAGCATTCATACCTGTAGGGATGTTCTGGGTTCTTTCCCTCCAGATAATCCAAGTTTCAATCACTGCCTCTGCTCCAGGGACTTTAGGAGGGGGAAGGGCCCCCGAGATACAGATCAGCACAGACTTTTCTTCTTTTCTTTCCGTAGCTAGAAGGAGAGAAGAGGCAGTGTCCTTGGCCATGGAGCTGCCGGTGGCCCCAGCCTGTCCCCACCTGACCTTGGGGGCAGAGAGGCTAAGGGACAGTGGAAGTGGTCCGCAGTGTGGTGAGCAGCCCCGGAGGAAGGGCACCGAGGAAGAGGAGGAGCTAGGAGGCCAGGTGCCCCTCCTCCTGCCATCTCTCCCCCTCTGGGCTCAGGTTGCAGAGAAGCCCGAGTGGTTCCTCACCCCTGTGCAGGACAGCCGAGCATCAGACACTGGTGATGGAGAAGCAGCCTGGGTTACAGTGCGAGATCAAGGTTATCCCCAGCTACCTAGTGAGACCCTCTCGCAGCATAAAGAGGTGAAGGTGGGGGAGGGGGGAATTATCAGGATCGGCATAAGTCAGGTGGCTCAGAGGGTGAAGTTGATGGCTGCGCAGGCCTGGCAAATTCAGTTCAACCCCCAGACTCCACAGAATATCGAAAATCCAGGCTTGGTGCAGCACTCCTTTAATCCCAGCACTTGAGAGGCAGAGGCAGGGGCTGGTGAGATGGCTCAGGGGTTAAGAGCACGGGCTGCTCTCCCAAAGGTCCTGAGTTCAAATCCCAGCAACCACATGGTGGCTCACAACCATCCGTAACAAGATTGGATGCCTCTTCTGGAGTGTCTAAGACAGCTACAGTGTACTGACATATAATAAATAAATAAATATTAAAAAAAAAAGAGAGAGGCAGAGGCAAGTGGATCTTTTGTGAGTTTGAGGCCAGCCTGATCTACATAGTGAATGCCAGGACAGCCAAGGCTATATAGAGACTCTGTCTCAAAAACAAAACAAAACAAAAATAAAACACCAAGAAGAACAAAATCTTGTTAGAATTAAAACTTTCCCCCAGTTGTGAGTGTTTTGCTTGCATATATGCCTGTGTATCGCATTCAGGCCTCATGCCTTTGGAGGTGAGAGGAGGGCGTTGGCGCTCCTGAGTCTGACTGCAGACAGTTGTGAGCTGCCCTGTACGCACTAGGAATAGAACCTGGGTCCTCTGCAATAGCAACAATTTCTCTAACCACTGAGCCATCTCTCTAACCCTCCCTGTATATTTTATTTATGTATATGTACATGCATAAGTTTATGTGTGTCTTGTCTGCTGAGACTGGAGTTACAAATGGTTGTGAGATGCCCTGTGGGTGCTGGGAACCAGACTAAGGTCTTCTAGGATCATCAAACCTTCTAACTACTGACTCCTTTCTCTGTGTAGCCCTGGCTGTCCTGGAACTCACTCGGTAGACCAGGCTAGCCTCAAACACAGATCCACCTGCCTCTGCCTCCAAGTGCTGGGATTAGAGGTGCATGCTACCCGCTGCCACCACCACTACCCAGATATTTTTCTTTAAAGGGTTATTAATGTGTACGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTAGTGTATTCCATGTGCAAACGGAGGTTTGAGGAGGCCCGGAGAGGTCAAATCCCCTGTAGCTGGAGTTACAGATGGTTTGTAAGCCACCCGATATGGGTGCTGGGAACCGAACTCTGGACCTCTAGAGAAGCCAGAAGTGCTCATGATCTCTGAGCTACCTCTCCAGTCTTCCTTCCCCTTTGTGTGGATTCCAGGGATGAACCCAGACCCTCAGACTCTGAGGACAGCACTTTTATCTGCTGAGTCATCTGCTGAGTCATCCCATCAGCTGCTCTTGTTTATTTTTGCCAGACTCCCTCTTACTTCATAACCAAAGCTGGCTTCAAATACAATCCTCCTGCTTCAGTCCCCCAAGCGCCGTGTTTCAGGAGTGTGTCACCCCCAGGCCTTACAAATCTCTGGCTTTCCCCCCTGGTAGGCAGGTTACCCCAGTTCAGGGAGGAATGGGGATGTGAGTTCGAAACACAGGTTAACTCTGATCGTACATATCCAGCCCTTCTCTTTCTGTTCCCAGCACAGCCATCAAACACAGCCTTGGGTAACAAGCCTGCCAAGGGCAGGCTCCCAAGCCCAGGCCCTGACTCCTAAGAACCATTAGCCCTTCTGTAATTCCTGCCAGGAGAGGTGCTATCTGTCTGGACCCCACCCGCACAGTTGCCTGGCAGCAGGCAGGTAGGCCTGGCACACTGTAAGAGGGGATGTTTGCCCCCTCCTCCCTCTCTTGCCCCCTTCCTTACTCCTCCTCCCCCTCTCTCCCCATTCCCTCACCCCTTCTCTCCATGTGGTCATGGCCGGCCTCTACTTCTCTGCTCTCTCCTTCTCTCTGCCTTTCTCTCCCTCTGCTACCCTCTTAACTCCCCTCCCCATGCTCTGAATAAACTCTATTCTATATTATACAGTCCTGTGGCTGGTCTCTCAGGGGGAAGGGATGCCTTGGCATGGGCCCGCTGAGACACCCCCTTTCCCCACACCCAGACAGAACATATCTTGATAGCTCTTTCTCCTTTTATGATCACAACACTGTCGACAGCTGCCTCTGATAGGCGTCCATCCCTCCTCCCTCCCAGTGCTGACCATGTATCATGTACCATGTACCATGTACCATGTACCATGTATCATGTATCATGTATCAGCTTCAGCAAGTTAATACTCTCACGCGCCATGGCCAGCGGCTTAGCCTGGCACCCTCAGGGTCCTGCCTGGCCCGCCTCACCAGCCTTCGAGGCTCATCTCCCAGGACCTGGGCATTTCTGCAGGCCCACCTGAGCGTCTTTCTCCATCTTGGCTCACACAGCTGCCTCTGCCTAACCAGGGCTCATTGCTTACGCCCCATGAGTGGAGGGAGCACTGACCCAAGGATGTACTCAGGTGAATCGTTTGTTCTTTGATAATTGTGCCAAGCCATGTTAAAACCAAAGCCTGCACAAGGCCCTTGATTGCACCCCCAGTGTCTCAAAAATAAAATAAGAAATCAAAGCTGGGTAGGGCAATAGCCTGTCTAGAATGATAGCACTCAAAGCAGAGGCAGGAAGATCCTGCAGTTTGTTTGAGACAGGATCTCACTATGTGACTCTGGCTGGCCTGGAATTGGCCTCAGACTTACAGAGATCCACCTGCCTCTGCCTCCTGAGTTTTGGAGTTTTATGTATTCATTTGTTTGTTTGTTTGTTTGTTTGTTTTTGAGATAGAGTCTCACCATGTAACCCTAACTAACAGAAATTCACCTGCCTCTGCCTCCCTAGTGTGTGCACCTCAGAGGCAGCAGTGGGAGGCTTCTGATTTTGAGGCCAGCCTAGGATACACAGTAAGACCCTGTCATTCCCCCTCCCCAAATAGTTTATGTAAATATATCCACCAGAAAAAAATGTTGAGGCCTAGGAGGGTGGCAAAAGGCTGGAGGCTGAGACGGAAAAATTACTCTAGCCCAGGGTTCAAGGCTAGACCAAGTGATATAGTAAGACTACAGCTCTAAAAAGTAAAACAAACAAAACAAAACAAAACAAAAAACTATAGTCAGGTAGCATGGTTGATCCATCCAAAGTAAAAGCAAGAAGACTAGAAGCTGACAAAGTGGCTCAGTGCGTGTGGCATGCGCCGTGTATCCTAACTGCGTGAGCTGGAGCTCCGGGACCCACAAGATGGAAGGAGAAAACTGACCCCCGAAAGTCGTCCTCTGACCGACCCCCACATGCAGACTGTGGCACATGAATACCCCTCCATACACACAAAGTAAATGTGTTTTAAAATAAAACGGGGTGTGGTGGCTTATACCTTGCAATCCTGGTACTAGGAAAGCTAAGCTGGGGAATTGTTATTTAGGGTCAGCTTGGGCTACATGTAATAGTTCTAGGCCAGCCTGGGCTATACAGCAAGACCTGTCTGAAAAAGCAAAATATCAATATTTCCCTGATAGAGGAAATACCATGATGAGGACTGTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGTGTATTTTCTTCTTTCTTTTTCTTTCTTAGGGGAGAGGTCCCTCCGTTGCACTACAGATGTGCTAGCCCCTGCAGGTTCCCCAGATTTGAAAAATAGGACTGCCGAATATTAGTTGTGGAACATTGTGATATTCCCCCATTAAAAATAAAGTAAAATAAAATTATGATACATAAGTGGGGAGGGGTGTGTGGCAGCAATGCATACCTTTAACGCCAGCACTCAGAGGCAGAGGCAGGTAGATCTCTGGGAGTTCAAGGCCAGTCTGGTCCATACAGTGAGTTCCAGAAATACACACAGAGAAATCCTGTCTCAAAAAACAAACAAAAAACAATATACATATATATGTATATGTATATATATATATATATATATTACATAAAATAGACAAATTAACTTAATTTGCTTTTGCCTGGCAGGGGTTGCTCATAACTGAGGAGTTTACCCCAAGGGACAAGCTGTTGAATAGGTCCCAGGCACATATATTTTCTCTTGACACCTAATTAATGGCTCTTTAGTCTTCACTTGCCAGGAGTCCTCCAGCTGATTCTAAGAAAACCCCTGTGTCTTCCTAAGTCTCTCTCCAGGGTCCCCTCCCAGGCCCATGATGACACCTGGAGAGAAGCCTGGAGCTTTGAGCTTCCAACATCCAACCATACAGCTCTGAGCACACCGGGTGGGGATGGGGAGAAAGTGAAAAACTATGGCCCACCAGGCTTTAACGTGCAAAGTCCAGCCTCCATGGCCAGGCCTTTCATCACAGCTACTTGAGAGATTGAGGCCAGAAGACTCAGAAGACTGTGAAGTTCAGGCTTCCCAAGCCACAGAGTGAGTCCAAGGCCAAGAGGAGCAACTTAATAAAACTATCTTGAAAGAGAGAGAGAGAAAGAAAGAGAGGGTGGGGAAGAGAGGGAGAGAGAAAGAGCAAAAGAAAGGAAAGGAAAGGAAAGGAAAAAGCTGGGGTTAAAGCTTAGAGCACCTGCCTAGAGTACCCCTGTAAAGGGCTGGGGGCATGGCTCAGTGGTAGAGTCCCTGCCTAGAATCCCCCAGGGAGGGGTTGGGGGCGTGGCTCAGGGGTAGAGCCCCTGCCTAGAATCCTCCAGTGAGGGGCTGGGGGCGTGGCTCAGGGGTAGAGCCCCTGCCTAGAATCCTCCAGTGAGGGGCTGGGGCATGGCTCAGGGGTAGAGCCCCTGCCTAGAATCCCCCATTGAGGGGCTGGAGGCGTGGCTCAGTAGCAGAGCCCCTGCCTAAAATGCCCCAGGGAGGGGGTGGGTGCCTCACTAGAGCACTTGGCTTATATTCAGGAAAAGAATTAAAATTTGAATAGGATCCTGGTGTGGTGACATAGGCCTTTCATCCCAGCATAGGCTTTTAATCTCTGGAGGCAGAGGTAGGCAGATTGCTGAATTCGAAGGCAGCCTAGTCTACGAATGCAGCCAGTTCCTCGACAGCCAGAGTTACACTGAGAAATCCTGCCCTGAAAAACAGAAATAAAACAAAACCCAACAACAGCAAAACCCCCAAAACCCAAAACAAAACAAAAACAAAAACCTAAGTAAACAAAATAATAACAGAAACCCCAACCAACCGAAGAAATAAAAACCTTGAATAGGAATCACGTGGAGTGTCTTTACAACTATAACCCAGATTCTGATCGTTGGTTCAGATGAGGTTTGAGGCCACAGGTCTAATAATGTGTTGATAATGGGCTCCACCCGAGATTGAGGGAAGCAGAGGGAATTCAGGTGGGAGGGTGGGCCATCGGTGATATAAGAACAGTGGTGTCAGCCTGCGATCGCGCCGCCACCATGAGGGAGATCGTGCTCACGCAGATCGGGCAGTGCGGGAATCAGATCGGCGCCAAGTTCTGGGAGGTGATCTCTGATGAACATGCCATCGACTCCGCTGGCACCTACCACGGGGACAGCCACCTGCAGCTGGAGCGCATCAACGTGTACTACAACGAGGCCAGCGGTGGCAGGTACGTGCCCCGCGCTGTGCTCGTGGATCTGGAGCCGGGCACCATGGACTCTGTGCGCTCGGGGCCCTTCGGGCAGGTCTTCAGGCCAGACAACTTCATCTTCGGTCAGTGTGGGGCCGGAAACAACTGGGCCAAGGGACACTACACCGAAGGCGCGGAGCTGATGGAGTCAGTGATGGACGTTGTCAGAAAGGAGGCTGAGAGCTGTGACTGCCTGCAGGGTTTCCAGCTGACCCACTCCCTGGGTGGGGGGACTGGGTCTGGGATGGGTACCCTTCTGCTCAGTAAGATCCGGGAGGAGTACCCAGACAGGATCATAAACACATTCAGCATCCTGCCCTCGCCCAAGGTGTCGGACACCGTGGTGGAGCCCTACAACGCCACCCTCTCAGTCCACCAGCTCATAGAAAACGCAGATGAGACCTTTTGCATAGATAACGAAGCTCTGTATGACATATGTTCCAAGACCCTAAAACTGCCCACACCCACCTATGGTGACCTGAACCACCTGGTGTCTGCTACCATGAGTGGGGTCACCACGTGCCTGCGCTTCCCGGGCCAGCTGAATGCTGACCTGCGGAAGCTGGCCGTGAACATGGTCCCGTTTCCCCAGCTGCATTTCTTCATGCCCGGCTTTGCCCCACTGACCAGCCGGGGCAGCCAGCAGTACCGGGCCTTGACTGTGGCTGAGCTTACCCAGCAGATGTTTGATGCTAAGAACATGATGGCTGCCTGTGACCCCCGTCACGGCCGCTACCTAACGGCGGCTGCCATTTTCAGGGGTCGCATGCCCATGAGGGAGGTGGATGAACAAATGTTCAACATTCAAGATAAGAACAGCAGTTACTTTGCTGACTGGCTCCCCAACAACGTAAAAACAGCCGTCTGTGACATCCCACCCCGGGGGCTAAAAATGTCAGCCACCTTCATTGGGAATAATACGGCCATCCAGGAACTCTTCAAGCGTGTCTCAGAGCAGTTTACAGCAATGTTCAGGCGCAAGGCCTTCCTCCACTGGTACACGGGCGAGGGCATGGATGAGATGGAATTCACCGAGGCCGAGAGCAACATGAACGACCTGGTGTCTGAATATCAGCAATATCAGGATGCCACGGCCGAGGAGGAGGAGGATGAGGAGTATGCCGAGGAGGAGGTGGCCGAACAAAAACTCATCTCAGAAGAGGATCTGTAGTTAATTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCACGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGACTCGAGTTATCCTGGGTGTCTGCTGAGCCAACAGTGGTAGTAAGGTAAGGGCAGGATGTGTCAAACTGCCAATAGAGAACTACTTACTCTTCAGGCTGAAGCTGATGGAACAGGTAACAAAGGCAAACACTAATCATGATCAGCAAGATGAAGCAGAAAGGGAACAAGGGGATATTAAATGTGTATAGACACGCTAGAGAGATGGCTCAGCAGTTAAGAGAACTAGCTGGTCTTTCAGAGGTCCTGAGATCAATTTTAGACACCCACATGGTGGCTCATGACCATCTATCTATAAATGGATCTGATTTTCATGTCTGGCAGTGTACAGAAGCTAACTGAAGAAAGGTGGAAGACCCACAAGAGTTCAAGATAAGCCCTATATAGTGAAGTTCAAGGCAAGCTTTTTCTACCTGAAACTTAGTCTCAAAAAAAAATGAATACGTAAACAGTCTTCCAGGGGATAAGAACCTTACAGAAAAAGCAGAAATGCCTGGGGCACTGGATTACCGATGTAATCAAATTCAGTCCTTGAATTGAACACAGGATTGCCTAGAGCAAGGCCAGCCAGAGATTCATCTCAGAGGGAGAAAGGTGTCTTTGGAGCAATTTTGTGGTAATCTAGTATGTATCACATAAGTTTAGACGCATTTGGGACTGGAAAGATGTGAACAAAGCACCCTATGGCTCACATCTGTCATTAACTCTAGTTCCAGTGCACCTGACACCGTCTTCTGGCCTCTGCAGTGACCAAGCACATGGGTAGTATGTAGACATATACATAAGCAAAACACACATCATTAAAAAGTGACATTTCCCAAAGGAAGCTGAAGAACCAGTTCTTGAGAAGATAGTAGAAATCAGAAGGGGAAATAGTAGACATACAGAGGGACTGACCAGGTTGTGTCACCTTTATAGGCTAGGCTAATGGATGATCGACACTAGCGCTCTTTGTGAAGGACACACAAATGAGACATAGTTTATAGGACTAAACACACTTCTAAGCAATTTAATGAGACTTAAGACCCTGTCTCTAGCAAATACTCTGGATGATATTCAGCTCAAGGCTCTTGTCAGACATGTTTCCATTTTCAAGGTGAGCTAACTGGCCAAAACTGCCAACAACCTGTAGTGAATAGAGAAGATGAGAAAATCTGGATTCTCAAATGACCTAATGAAAGCCACTGGAGCGCCATATGGTTTCTGTGAAAATGCCTTTTCAATCATTAACCTCTTAAATGAGTGTTAGCATCCTAACTAATGAGTGGTGCAGAATAGTGGGTCTGCTTAGCTTAACTAAGGCCAAGAAAACAAAACAGGAAATTCATTCCATGTCATGAGACTCATACTACGAGGTTCCCTTAGACCTCAGGAGAAAAAGTCTTTGGCTGTAAGAACACACCTCAGTGGATGTGGTAGACTATGCCTTTACTCTTTTTTTTTTTTTTTTTTTTTTTTGGGTTTTTTCGAGACAGGGTTTCTCTGTGTAGCCTTGGCTGTCCTGGAACTCACTTTGTTGACCAGGCTGGCCTCGAACTCAGAGATCCGCCTGCCTCTGCCTCTCAAGTGCTAGAATTAAAAGTGTGCGCCACCACTCCCAGCTATATAGACTATGCCTTTAATCAGGACACTTGGGAGGCAGGGGGATCTCAAATTCAATTCCAGACAGATGACTGACAAACACACACATACACCATTCGTCTTTTCTTTTTTTCTTTTTTCTTTTGAAAACAGGTTTCTCTATGCAGCCCTGGCTTTCCTAGAATTCAATCTATAGACCCAGCTGGCCTCAAACACAGAACCTCTTGCCTCTGCCTCCTCAACACTATGACTAAAGGTGTGAACCACCACCACCACCACCCACCTGGCCAAAGAATGAATTGAGTGAATAAGTACGAGTGCAACTCTCCAGCAGCCTGAGGCAAGAAAGCTTAACAGTTGAACCTGAGACCCTACTTCGTTTGTGTTGTACATATCTTCTCATAATGAACTAGGCAGCCTAGTCTCTCTACCAAATACACCATGCACTTACACCTCCTCAAACCAA-3'。

an embodiment of the present application also provides an hTUBB8 gene editing system comprising the donor fragment described above.

In some of these embodiments, the hTUBB8 gene editing system further comprises one or more of a Cas9 nuclease, a Cas9mRNA, and a gRNA.

In some of these embodiments, the nucleotide sequence of the above gRNA is shown as SEQ ID NO. 1.

Specifically, the nucleotide sequence shown in SEQ ID NO. 1 is 5'-GAACACTAGTGCACTTATCCTGG-3'.

It is understood that the gRNA targeted mutation site is a safe site H11 region (region between 4.5kb downstream of Drg gene and 0.7kb upstream of Eif4enif1 gene), and that insertion of foreign gene into this region does not affect expression of other genes in mice.

An embodiment of the present application also provides a recombinant cell comprising the above donor fragment targeting the hTUBB8 gene or the above hTUBB8 gene editing system.

It is understood that the hTUBB8 gene of the cell is edited by the hTUBB8 gene editing system, and the obtained recombinant cell can transmit the mutated genotype to the daughter cell by means of cell division, so that the recombinant cell has stability and heritability.

The embodiment of the application also provides a construction method of the ovum maturation disorder animal model, which comprises the steps S10 to S50.

Step S10: the gRNA and donor fragment targeting the hTUBB8 gene were designed against the hTUBB8 c.785g > a mutation site of the target animal.

In one specific example, the nucleotide sequence of the donor fragment targeting the hTUBB8 gene is shown in SEQ ID NO. 2.

By adopting the donor fragment of the specific hTUBB8 gene and combining with the Crispr/Cas9 system, the mutation site of the hTUBB8 gene can be specifically knocked into the genome of a target animal, so that an animal model carrying the mutation of the hTUBB8 gene is constructed.

Step S20: transferring the hTUBB8 gene editing system into fertilized eggs to obtain fertilized eggs transferred into the hTUBB8 gene editing system.

In some of these embodiments, the hTUBB8 gene editing system described above includes the donor fragment, gRNA, and Cas9mRNA described above.

In some of these embodiments, the method of transferring is microinjection.

Step S30: the fertilized egg in step S20 is transplanted into a female animal and F0 generation is produced.

Step S40: mating the F0 generation in the step S30 with a wild type to obtain an F1 generation heterozygote, selfing the F1 generation heterozygote, and screening out a homozygous F2 generation serving as an ovum maturation disorder animal model.

In some embodiments, the target animal comprises a rat or mouse.

Alternatively, the target animal is a mouse.

In some embodiments, the above construction method further includes step S50.

Step S50: and (5) identifying the genotype of the target animal.

In some of these embodiments, primer pairs having nucleotide sequences shown in SEQ ID NOS.4-7 are used to identify the genotype of the target animal.

In some embodiments, the step of identifying the gene in the mouse comprises step a, step b, step c, step d, and step e.

Step a: genomic DNA of the toe and/or tail of the mice was extracted.

Step b: and taking genome DNA extracted from the toe and/or tail of the mouse as a template, and respectively adopting a first amplification primer pair with a forward primer sequence shown as SEQ ID NO. 4 and a reverse primer sequence shown as SEQ ID NO. 5 and a second amplification primer pair with a forward primer sequence shown as SEQ ID NO. 6 and a reverse primer sequence shown as SEQ ID NO. 7 for PCR amplification to obtain an amplification product.

Specifically, the first amplification primer pair has the sequences: the forward primer hTUBB8-F3 (SEQ ID NO: 4): 5'-CTCTACTGGAGGAGGACAAACTG-3'; the reverse primer hTUBB8-R3 (SEQ ID NO: 5): 5'-GTCTTCCACCTTTCTTCAGTTAGC-3'; a second amplification primer pair having the sequence: the forward primer hTUBB8-F10 (SEQ ID NO: 6): 5'-GCCTCCAAGTCTTGACAGTAGATT-3'; reverse primer hTUBB8-R10 (SEQ ID NO: 7): 5'-TGAAAACCTCCAGAGCCCTTTC C-3'.

In some of these embodiments, the method of identifying comprises one or more of PCR and Sanger sequencing.

In some of these embodiments, in step b, the amplification procedure of the PCR is set to: pre-denaturation at 95 ℃ for 5min; denaturation at 94 ℃ for 30-45 s, annealing at 56-72 ℃ for 30-60 s, extension at 72 ℃ for 30-60 s, and co-circulation for 30-35 times; finally, the PCR amplification product is extended at 72 ℃ for 5 to 10 minutes and stored at 4 ℃. It will be appreciated that in some other specific examples, the PCR procedure may be rationally adjusted.

In one specific example, the amplification procedure for PCR is set to: pre-denaturation at 95℃for 5min, denaturation at 94℃for 30s, annealing at 60℃for 30s, extension at 72℃for 30s for 35 cycles, extension at 72℃for 5min, and storage of PCR amplified products at 4 ℃.

Step c: agarose gel electrophoresis was performed on the PCR amplified products.

In one embodiment, in the step c, 3-5 mu L of PCR amplification product and DL5000 DNA Marker are respectively taken and put into a gel hole, and electrophoresis is carried out for 10-20 min at 220V, and imaging and photographing are carried out.

In one embodiment, in step c, the band size of the PCR amplification product is determined by agarose gel electrophoresis.

Step d: sequencing the amplified product.

In one embodiment, in step d, the sequencing method is Sanger sequencing.

In one embodiment, in step d, the sequencing analysis is performed using the same primer pair as the PCR amplification primer pair.

Step e: the hTUBB8 genotype of the mice was identified.

In one embodiment, in step e, the sequence obtained by sequencing step d is identical to the corresponding DNA sequence of the wild-type hTUBB8 gene as set forth in SEQ ID NO:8, and analyzing whether the mice are hTUBB8 knockout mice.

Specifically, the nucleotide sequence shown in SEQ ID NO. 8 is: 5'-CTCTACTGGAGGAGGACAAACTGGTCACTTTTCAGCAAAACCTGGCTGTGGATCAGGGCAGTCTGGTACTTCCAAGC TCATTAGATGCCATCATGCTCTCACTGCCTCCTCAGCTTCAAGAGGAATCTGGAAAAAGCAGTCCCACTGGTCAGGAAAGGAACACTAGTGCACTTATCCTGGGTGTCTGCTGAGCCAACAGTGGTAGTAAGGTAAGGGCAGGATGTGTCAAACTGCCAATAGAGAACTACTTACTCTTCAGGCTGAAGCTGATGGAACAGGTAACAAAGGCAAACACTAATCATGATCAGCAAGATGAAGCAGAAAGGGAACAAGGGGATATTAAATGTGTATAGACACGCTAGAGAGATGGCTCAGCAGTTAAGAGAACTAGCTGGTCTTTCAGAGGTCCTGAGATCAATTTTAGACACCCACATGGTGGCTCATGACCATCTATCTATAAATGGATCTGATTTTCATGTCTGGCAGTGTACAGAAGCTAACTGAAGAAAGGTGGAAGAC-3'.

In one embodiment, in step e, the knock-in mouse acquires the hTUBB8 (c.g785a) gene mutation.

The method for constructing the ovum maturation disorder animal model has at least the following advantages:

(1) An animal model of egg maturation disorder in which egg maturation is impaired was constructed by specifically knocking in the hTUBB8 (c.g785a) gene mutation by targeting the donor fragment of the hTUBB8 gene and targeting the gRNA of the hTUBB8 gene.

(2) The construction method can realize the hTUBB8 (c.G785A) gene knock-in of specific time and space through conditional gene knockout.

In addition, an embodiment of the present application also provides a method for screening or identifying a drug for treating an egg maturation disorder, which uses the egg maturation disorder animal model prepared by the above-mentioned egg maturation disorder animal model construction method to screen or identify a drug for treating an egg maturation disorder.

The method for screening or identifying the medicines for treating the egg maturation disorder utilizes the egg maturation disorder animal model prepared by the construction method of the egg maturation disorder animal model to screen or identify the medicines for treating the egg maturation disorder, and the method is more accurate for screening or identifying the medicines related to the egg maturation disorder because the egg maturation disorder animal model is used for generating disorder for egg maturation.

The present application will be further described with reference to specific examples and comparative examples, which should not be construed as limiting the scope of the present application. The materials used in the following examples were all commercially available, unless otherwise specified, the equipment used, and the processes involved, unless otherwise specified, were all routinely selected by those skilled in the art.

Example 1

1. Applicants have found clinically an example of a patient carrying a mutation in the TUBB8 gene, which patient has a mutation in the TUBB8 gene of c.G785A.

TUBB8 is a subtype of beta-tubulin, distributed only in egg cells and early embryos, not expressed in somatic and sperm cells, and the gene expresses beta-tubulin. TUBB8 is specifically expressed in oocytes and is the predominant form of tubulin constituting the spindle B of oocytes. TUBB8 mutations affect folding of the corresponding protein, alter the dynamics of microtubule action, cause a disturbance in spindle shape and structure, and in turn affect meiosis, leading to oocyte maturation disorders, and other phenotypes.

2. Sequence design

The gRNA targeting site designed for inserting exogenous hTUBB8 (c.G785A) gene into mouse is the safe site H11 region (the region between 4.5kb downstream of Drg gene and 0.7kb upstream of Eif4 end 1 gene), the insertion of exogenous gene into this region will not affect the expression of other genes in mouse, the nucleotide sequence of gRNA is: (SEQ ID NO: 1) 5'-GAACACTAGTGCACTTATCCTGG-3'. Since the TUBB8 gene is a gene unique to primate, there is no expression of homologous genes in mice, and the ZP3promoter of mice is used to drive expression. The mouse ZP3promoter is a well-defined sequence promoter that has been reported to initiate gene expression in oocytes of primary follicles, peak expression in oocytes of growing follicles, and is consistent with the pattern of initiation of expression of human TUBB8 in oocytes of growing follicles. The present invention therefore selects the mouse ZP3promoter to initiate specific expression of the exogenous hTUBB8 (c.g785a) gene in the primary follicular developmental stage of the mouse to achieve a pattern that mimics the expression of the TUBB8 (c.g785a) gene in the patient as completely as possible. That is, in order to simulate the characteristic of the time-specific expression of the hTUBB8 (c.g785a) gene in the ovum of a patient, the present application uses the mouse ZP3promoter to drive the expression of the hTUBB8 (c.g785a) gene, and the Donor DNA sequence is designed as follows: the nucleotide sequence of the Mouse Zp3 master-Kozak-Human TUBB 8-variant CDS-Myc tag-WPRE-BGH pA is shown as SEQ ID NO. 2, and all the Donor DNA sequences are obtained through artificial synthesis.

3. Microscopic injection of F0 generation mice

Designing and synthesizing gRNA aiming at a specific site of the H11 region, and in-vitro transcribing the gRNA and the Cas9 nuclease into mRNA, and designing and synthesizing a Donor DNA template (SEQ ID NO: 2) with high homology with the immediately upstream and downstream sequences of the specific target; gRNA, donor DNA and Cas9mRNA are injected into fertilized eggs of a C57BL/6J mouse through microinjection, the microinjected fertilized eggs are transplanted into the uterus of a female mouse, and a born mouse is an F0 generation mouse.

4. Propagation of F1 mice

Mating and breeding the F0 generation mice with positive sequencing verification with WT wild mice to obtain genetically stable F1 generation heterozygous hTUBB8 (c.G785A) gene knock-in mice.

5. Propagation of F2 mice

The heterozygous mice of F1 generation were bred by mating with each other to obtain the homozygous mutant mice of F2 generation, namely the knock-in mice model carrying the mutation of hTUBB8 (c.G785A), as shown in FIG. 1.

6. Mouse identification

After the 6.1F0 generation, F1 generation and F2 generation mice are born for 10 days, the alkali lysis method is used for extracting the mouse DNA, and the specific steps are as follows:

(1) The toe or tail of the mice was cut in the animal house and the cut tissue was brought back in a 1.5mL EP tube, 180 μl of 50mM NaOH was added and the tissue was minced using alcohol sterilized scissors.

(2) The EP tube was inserted into the float and placed in a 96 ℃ metal bath for heating for 30min, during which time mixing was reversed 1 time.

(3) mu.L of 1mM Tris-HCl was added thereto, and the mixture was thoroughly inverted and mixed.

(4) Centrifuge at 12000rpm for 2min, aspirate the supernatant into a new EP tube and mark.

The offspring mice bred by hybridization were genotyped based on toe genomic DNA by PCR and Sanger sequencing using primers comprising: a first amplification primer pair having the sequence: the forward primer hTUBB8-F3 (SEQ ID NO: 4): 5'-CTCTACTGGAGGAGGACAAACTG-3'; the reverse primer hTUBB8-R3 (SEQ ID NO: 5): 5'-GTCTTCCACCTTTCTTCAGTTAGC-3'; a second amplification primer pair having the sequence: the forward primer hTUBB8-F10 (SEQ ID NO: 6): 5'-GCCTCCAAGTCTTGACAGTAGATT-3'; the reverse primer hTUBB8-R10 (SEQ ID NO: 7): 5'-TGAAAACCTCCAGAGCCCTTTC-3'; the reaction system of PCR amplification is shown in Table 1,

TABLE 1

Reagent name	The volume added
		Green Master Mix	15μL
gDNA	1.5μL
		hTUBB8-F3/F10(10μM)	0.5μL
hTUBB8-R3/R10(10μM)	0.5μL
		RNase Free dH ₂ O	12.5μL
Total	30μL

The PCR reaction system solution was mixed uniformly and then put into a PCR instrument for amplification, and the amplification procedure is shown in Table 2.

TABLE 2

Temperature (temperature)	Cycle number
		95℃(5min)	1
94℃(30s)→72℃(30s)→Tm℃(30s)→72℃(30s)	35
		72℃(10min)	1
4 ℃ (preservation)

And (3) carrying out agarose gel electrophoresis on the PCR amplified product, determining that the amplified target band is correct, and then sending the rest PCR amplified product to the department of Praeparata Biotechnology, inc. (Beijing, china) for Sanger sequencing verification.

6.2HE staining identification

The method comprises the following specific steps:

(1) After washing the ovarian samples 2 times in PBS, they were fixed overnight at 4 ℃ in 4% pfa.

(2) The sample was placed in an embedding medium OCT, frozen at-20 ℃ overnight, sliced in a frozen microtome, and mounted on a slide.

(3) Immersing a sample slide in clear water for 1min to wash OCT, immersing the sample slide in a dye vat containing hematoxylin for 3-5min, and then immersing the sample slide in tap water for three times, each time for 5min.

(4) Immersing the slide glass in absolute ethyl alcohol for 2 times each for 2 seconds, immersing in a staining jar containing eosin for 1-3min, and immersing in absolute ethyl alcohol for three times each for 5min.

(5) The slide was soaked in the transparentizing agent three times for 2min each.

(6) The tablet is sealed and photographed by microscopic observation.

7. Analysis of results

(1) Sequencing result analysis and sequence comparison

The application successfully constructs a Mouse (C57 BL/6) model containing the gene knock-in of hTUBB8 (c.G785A) by using a Crispr-Cas system, as shown in figure 1, wherein Mouse ZP3promoter represents a Mouse ZP3promoter region, kozak-Human TUBB8 (c.G785A) CDS-Myc tag represents a region which sequentially comprises a Kozak sequence, an hTUBB8 (c.G785A) CDS sequence and a Myc tag sequence from a 5 'end to a 3' end, WPRE is a cis-acting RNA element which is a post-transcriptional regulatory sequence, BGH pA represents a transcription termination signal region, and arm represents a homology arm region.

1) The genotyping result judgment criteria are shown in table 3 below:

TABLE 3 Table 3

2) Analysis of results of genotypes of mice

(1) Wild type: the PCR procedures in Table 2 were carried out using the primers hTUBB8-F3, hTUBB8-R3 and hTUBB8-F10, respectively, in accordance with the system of Table 1, whereby a band of 519bp was obtained in the PCR products of the primers hTUBB8-F3 and hTUBB8-R3, respectively, the agarose gel electrophoresis chart was shown in FIG. 2, the wild-type mouse PCR amplification product electrophoresis chart was shown in lane WT, and Sanger sequencing was carried out on the PCR products using the primers hTUBB8-F3 and hTUBB8-R3, respectively, as shown in SEQ ID NO: 8.

Specifically, the nucleotide sequence shown in SEQ ID NO. 8 is: 5'-CTCTACTGGAGGAGGACAAACTGGTCACTTTTCAGCAAAACCTGGCTGTGGATCAGGGCAGTCTGGTACTTCCAAGCTCATTAGATGCCATCATGCTCTCACTGCCTCCTCAGCTTCAAGAGGAATCTGGAAAAAGCAGTCCCACTGGTCAGGAAAGGAACACTAGTGCACTTATCCTGGGTGTCTGCTGAGCCAACAGTGGTAGTAAGGTAAGGGCAGGATGTGTCAAACTGCCAATAGAGAACTACTTACTCTTCAGGCTGAAGCTGATGGAACAGGTAACAAAGGCAAACACTAATCATGATCAGCAAGATGAAGCAGAAAGGGAACAAGGGGATATTAAATGTGTATAGACACGCTAGAGAGATGGCTCAGCAGTTAAGAGAACTAGCTGGTCTTTCAGAGGTCCTGAGATCAATTTTAGACACCCACATGGTGGCTCATGACCATCTATCTATAAATGGATCTGATTTTCATGTCTGGCAGTGTACAGAAGCTAACTGAAGAAAGGTGGAAGAC-3'.

(2) Homozygous type: the PCR procedure in Table 2 was performed using the primers hTUBB8-F3, hTUBB8-R3 and hTUBB8-F10, respectively, according to the system in Table 1, to obtain a PCR product of the primers hTUBB8-F10 and hTUBB8-R10 having a 748bp band, the agarose gel electrophoresis pattern of which is shown in FIG. 2, lane M: marker (5000 bp), sanger sequencing was performed on PCR products using hTUBB8-F10, hTUBB8-R10 primers, and the results were shown in SEQ ID NO. 9.

Specifically, the nucleotide sequence shown in SEQ ID NO. 9 is:

5'-GCCTCCAAGTCTTGACAGTAGATTATAATCCTTCAGCTGCCCACTCTACTGGAGGAGGACAAACTGGTCACTTTTCAGCAAAACCTGGCTGTGGATCAGGGCAGTCTGGTACTTCCAAGCTCATTAGATGCCATCATGCTCTCACTGCCTCCTCAGCTTCAAGAGGAATCTGGAAAAAGCAGTCCCACTGGTCAGGAAAGGAACACTAGTGCACGGCGCGCCAATTCCTTTTAGCCCCGGTGGGGCAGGTGATTAGGAGCTGTGAAGCCTTTGTTCTTATTGGCAAAAGGGTCCCATATTCTTTCCCTTATACTTGTTGCCATATTAAATGGGTAATGTTTATAAAAGGTTTCCCCAGTGTCTGGCCCGCTCTAATGTGCTTAGCTATTGCTATGACTGCCCCCGTCTGACCTCCAAGACAAGAGCCAGATCTCATTCAATAGTTGTACGTGGCATATGATGTTCAGAAAATGTTTGTTGATGGGCTGAGTGAAATTGTGGCTATGAAGTCATAGAATGAAACAGAGGGGTCTCAGGAAGTAGTCAATGAGTTGGCTCAGCAGGTAAAGGTGACTGCTGCCAAGTCTGATGACCTAAGTTCAATTCCTGGGACCCAAATGTCAGAAGGCAAAACACTCTTGCAAGTTGCCCTGTGACTGTCACTTGTGCATGCCTGCACACACATAAATAAATAAATGCAATATTAAGAATTTTTTTTTTTTTAAAGAAAGGGCTCTGGAGGTTTTCA-3'。

(3) heterozygous: the PCR procedures in Table 2 were carried out using the primers hTUBB8-F3, hTUBB8-R3 and hTUBB8-F10, respectively, in accordance with the system of Table 1, and the results are shown in FIG. 2, whereby PCR products of the primers hTUBB8-F3, hTUBB8-R3 and hTUBB8-F10, and hTUBB8-R10 were obtained, and bands were formed at 519bp and 748 bp.

(2) HE staining identification

As shown in FIG. 3, HE-stained hTUBB8 (c.G785A) gene knockin mice had smaller follicular sizes than wild-type mice of the same period.

In summary, the Donor DNA sequence designed in the present application is: the Mouse Zp3promoter-Kozak-Human TUBB 8-variant CDS-Myc tag-WPRE-BGH pA comprises a mutation site of TUBB8 (c.G785A) gene carried by a patient, and the expression of the gene is started in a primary follicular stage of an oocyte by a ZP3promoter, so that an effective Mouse model capable of simulating the mutation site and expression characteristics of the TUBB8 (c.G785A) gene carried by the patient is constructed. The heterozygote mice are bred later, and homozygous mice knocked in with the gene of the TUBB8 (c.G785A) can be continuously prepared and used as tool mice for subsequent TUBB8 gene function experiments. The method has important significance in the aspects of deeply analyzing the pathogenicity of the mutation, exploring the treatment strategy, providing theoretical basis for the treatment of patients with the gene defects clinically, and the like.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples only represent a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the claims. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present application is to be determined by the claims appended hereto.

Claims

1. A donor fragment targeting the hTUBB8 gene, wherein the donor fragment comprises, in order from the 5 'end to the 3' end, a ZP3promoter region, a Kozak sequence, a mutated hTUBB8 gene fragment, a tag sequence, a WPRE sequence, and a terminator sequence, wherein the mutated hTUBB8 gene fragment has the following mutations compared to the wild-type hTUBB8 gene: c.785G > A, the cDNA sequence of the wild hTUBB8 gene is shown in SEQ ID NO: 3.

2. The donor fragment targeting the hTUBB8 gene according to claim 1, wherein the nucleotide sequence of the donor fragment is set forth in SEQ ID No. 2.

3. An hTUBB8 gene editing system, comprising the donor fragment of claim 1 that targets the hTUBB8 gene;

4. A recombinant cell comprising the donor fragment of any one of claims 1-2 that targets the hTUBB8 gene or the hTUBB8 gene editing system of claim 3.

5. The construction method of the ovum maturation disorder animal model is characterized by comprising the following steps:

constructing an ovum maturation disorder animal model using the hTUBB8 gene editing system of claim 3.

6. The method for constructing an animal model for egg maturation disorder according to claim 5, comprising the steps of:

7. The method of claim 6, wherein the transferring comprises microinjection.

8. The method for constructing an egg maturation disorder animal model according to any one of claims 6 to 7, further comprising the steps of:

9. The method of claim 8, wherein the method of identifying comprises one or more of PCR and Sanger sequencing.

10. A method for screening or identifying a drug for treating an egg maturation disorder, characterized in that the drug for treating an egg maturation disorder is screened or identified using the egg maturation disorder animal model produced by the method for constructing an egg maturation disorder animal model according to any one of claims 5 to 9.