CN119913206B - Construction method and application of Kaimachia rat model - Google Patents

Abstract

The invention discloses a construction method and application of a Kaimachia rat model, and belongs to the technical field of animal models. The construction method of the Kaimachia rat model is to mutate the 38 th nucleotide of the ATCAY gene exon 3 of the rat from G to A. The rat model of the Kaman ataxia constructed by the invention provides a research model tool for researching the function of ATCAY genes in the Kaman ataxia and screening or preparing medicines for preventing or treating the Kaman ataxia.

Description

Construction method and application of Kaimachia rat model

Technical Field

The invention belongs to the technical field of animal models, and particularly relates to a construction method and application of a Kaimachia rat model.

Background

The Kaman ataxia belongs to autosomal recessive genetic diseases, and the patients have symptoms of dystonia, ataxia gait, trunk ataxia and the like from birth. Researchers have located mutations in the ATCAY gene of human 19p13.3 at this cause, and so far have found four mutations, including one missense mutation, one splice mutation and two frameshift mutations. The ATCAY gene encodes a caytaxin protein which is only present in neuronal tissue and is highly expressed at the presynaptic site of gabaergic neurons, and the inclusion of homologous domains which are highly conserved across species is thought to be closely related to the function of the caytaxin protein, but no research system has elucidated the function of the caytaxin protein in the nervous system of the body so far.

Animal models are the main tools for researching human pathogenesis, screening drug therapeutic targets and developing therapeutic means. The gene editing animal model can simulate the disease phenotype and pathological characteristics of human more accurately, and has very wide research and application prospects. The current animal model for ATCAY gene mutation comprises the following four types of mouse model with 3 # exon B1 element inserted into jittery, mouse model with 1 # intron IAP element inserted into hesitant, mouse model with 5 # intron with 2 bp deletion sidewick and BNIP-H knockdown zebra fish model. However, the above animal model focuses on dystonia caused by gene mutation, and cannot completely simulate the human disease phenotype of the keamateur ataxia, so that the technical problem of how to obtain an animal model for researching pathogenesis of the keamate ataxia and providing a disease for drug screening is needed to be solved in the technical field.

Disclosure of Invention

Aiming at the problem that the existing ATCAY gene mutant animal model can not completely simulate the Kaman ataxia phenotype, the invention aims to provide a construction method of a rat model which is more in line with the Kaman ataxia phenotype. The invention also aims to provide application of the constructed rat model of the ataxia.

The aim of the invention is achieved by the following technical scheme:

the invention provides a construction method of a rat model with the ataxia of the Kalman, which comprises the steps of enabling a 38 th nucleotide of a ATCAY gene exon 3 of a rat to be mutated from G to A, namely, c.115G > A mutation of a ATCAY gene of the rat and G39S mutation of encoded protein.

In some embodiments, the method of mutating nucleotide 38 of the ATCAY gene exon 3 of the rat from G to a comprises gene editing techniques such as CRISPR/Cas9, ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), and the like, preferably using CRISPR/Cas9 gene editing techniques.

In some embodiments, the method for constructing a model of a rat with ataxia, comprising the steps of:

(1) Active sgrnas, cas9 mRNA were obtained by in vitro transcription.

(2) Mixing the active sgRNA, cas9 mRNA and a donor oligonucleotide sequence, microinjection into a fertilized egg of a rat, and transplanting the fertilized egg into a oviduct of a surrogate rat for inoculation to obtain an F0 generation rat;

(3) The F0 generation positive rat is hybridized with a wild rat to obtain an F1 generation ATCAY gene heterozygous mutant rat, the F1 generation ATCAY gene heterozygous mutant rat is selfed to obtain an F2 generation ATCAY gene homozygous mutant rat, and the Kasman ataxia rat model is obtained.

In step (1), the sequence of the sgRNA is preferably GGATCACCTGGGCGGCACGG-TGG (SEQ ID NO. 1).

In step (2), the sequence of the donor oligonucleotide is preferably ACCGCTCCCAGAAGACACCGGGGAGGATCACCTGGGCAGCACGGTGGAAGACTCCTCCT (SEQ ID NO. 2).

In step (3), the mutation of the rat ATCAY gene is preferably detected by PCR using the following primers:

5- 'TTCAATGCATTCCTCCTGCT-3' (SEQ ID NO. 3);

The downstream primer is 5- 'TCTTACCCTTGTTGGCCTTG-3' (SEQ ID NO. 4).

In the method for constructing the Kaimachia rat model, the rat is preferably an SD rat.

The Kaomaxie rat model obtained by the construction method shows the phenotype that the sick rat is ill 1 to 2 weeks after birth, has obvious difference from the normal rat in morphology and serious phenotype, and has the survival period not exceeding 1 month of age without being assisted by artificial feeding. The upper and lower bodies of the sick mice are reversely twisted, high-frequency tremors appear in limbs, obvious dystonia is accompanied, normal crawling and autonomous feeding cannot be realized, and three times of manual feeding are required.

The invention also provides a targeting system for constructing a Kasman ataxia rat model, wherein the Kasman ataxia rat model is ATCAY:115G > A gene editing rat, and the targeting system comprises sgRNA, donor oligonucleotide and Cas9 mRNA.

The invention also provides application of the rat model obtained by the construction method in research of the ataxia disease, and the application aims at treatment of non-disease. The research is research on pathogenesis of the Kaemann ataxia disease, mechanism research or screening of medicines for preventing or treating the Kaemann ataxia disease.

The invention also provides application of the rat model obtained by the construction method in screening or preparing a medicine for preventing or treating the Kaschin ataxia, wherein the application aims at the treatment of non-diseases.

Compared with the prior art, the invention has the advantages that a novel strain of the rat with the function loss of the ATCAY gene coding protein Caytaxin and the Kaschin ataxia phenotype caused by ATCAY:115G > A point mutation is obtained for the first time, and a research model tool is provided for researching the function of ATCAY genes in the Kaschin ataxia and screening or preparing medicines for preventing or treating the Kaschin ataxia.

Drawings

FIG. 1 is a schematic diagram of a target strategy for point mutation of caytaxin protein-encoding gene ATCAY. The genomic region at position ATCAY is schematically shown and the solid bars represent Open Reading Frames (ORFs).

FIG. 2 is a diagram showing the genotyping and sequencing of ATCAY gene point mutant gene-edited rat according to the present invention. 5. 7, 19 and 31 are respectively the serial numbers Founder, 7, 19 and 31 of the first-generation mice, WT is a wild rat, a is a sequencing peak diagram of a F0-generation first-generation mice Founder 5 and PCR product, black arrows indicate base mutation sites, b is a base sequence of F0-generation first-generation mice Founder, 7, 19 and 31, and c is an amino acid sequence of F0-generation first-generation mice Founder 5, 7, 19 and 31.

FIG. 3 is a diagram showing phenotype examples of ATCAY gene-editing homozygous mutant rats and heterozygous mutant rats of the present invention. a is F2 generation ATCAY gene editing homozygous mutant rat, which is ill after one to two weeks of birth, and the phenotype of the ill rat is obviously different from that of normal rat. The upper and lower bodies of the sick mice are twisted in opposite directions, the limbs are tremor at high frequency, dystonia, incapability of crawling normally and eating spontaneously, manual feeding is needed three times a day, the situation of the Kasman ataxia phenotype is met, b is F2 generation ATCAY gene editing heterozygous mutant rats, and the bodies of the rats are free from twisting, the limbs are free from tremor, and the rats can crawl normally and have eating capability, and the phenotype is different from that of normal rats.

Detailed Description

According to the invention, an SD rat is accidentally found to have reverse twisting of the upper half body and the lower half body within 1 week after birth, and has myotonic phenotypes such as high-frequency tremor and the like on limbs, and the 38 th nucleotide of exon 3 of ATCAY gene of the SD rat is found to be mutated into A (ATCAY: c.115G > A) from G after full genome sequencing of the SD rat, which corresponds to the Kaman ataxia caused by mutation of ATCAY gene of human beings. Then, ATCAY:115G > A point mutation rats are further constructed by a gene editing means, and the constructed rats are found to have reverse distortion of the upper and lower bodies of the rats after 1 to 2 weeks of birth, with limb high-frequency tremors, unable to normally crawl and independently eat, and have an ataxia phenotype which requires three times of manual feeding per day, so that the Kaman ataxia rat model can be obtained by the gene editing means that the c.115G > A mutation of the ATCAY gene of the rats is generated.

The following will describe embodiments of the present invention in detail by referring to examples, so that the implementation process of how the present invention applies technical means to solve technical problems and achieve technical effects can be fully understood and implemented.

Example 1

(1) According to the mutation site of the self-mutation SD rat ATCAY gene with the phenotype of reverse twisting of the upper and lower body, high-frequency tremor of limbs and the like, sgRNA and donor oligonucleotides (see below in sequence) are designed, and a CRISPR/Cas9 technology is utilized to construct a point mutation rat of ATCAY gene exon 338 from G to A (shown in figure 1), namely ATCAY:c.115G > A.

The sequence near the target of the wild-type allele before mutation is ACCGCTCCCAGAAGACACCGGGGAGGATCACCTGGGC (GGC) ACGGTGGAAGACTCCTCCT (SEQ ID NO. 5). The sequence near the target of the mutated wild-type allele is ACCGCTCCCAGAAGACACCGGGGAGGATCACCTGGGC (AGC) ACGGTGGAAGACTCCTCCT (SEQ ID NO. 6). After successful mutation, the sequence in the wild-type allele brackets will be replaced by the sequence in the mutant allele brackets.

The sgRNA sequence GGATCACCTGGGCGGCACGG-TGG (SEQ ID NO. 1).

The donor oligonucleotide sequence ACCGCTCCCAGAAGACACCGGGGAGGATCACCTGGGCAGCACGGTGGAAGACTCCTCCT (SEQ ID NO. 2).

(2) Mixing two single-stranded oligonucleotides sgRNA, annealing for 5min at 95 ℃, naturally cooling to room temperature to form double chains, and connecting the formed double chains of the sgRNA with a linearized pGK1.1 vector under the action of T4 DNA ligase to construct the sgRNA expression vector pGK1.1-sgRNA. Subsequently, the recombinant plasmid was transformed into DH 5. Alpha. Competent cells, and the correctly inserted recombinant plasmid was identified by screening positive clones containing the kanamycin resistance gene and sequencing in combination with the target DNA. After correct colony clones are selected, amplification culture is performed, plasmid DNA is extracted, and templates are prepared for in vitro transcription. Further active sgrnas are obtained by in vitro transcription.

(3) Cas9 expression plasmid (Addgene No. 44758) obtains Cas9 mRNA by in vitro transcription.

(4) The donor oligonucleotide sequences were synthesized directly by the company and the obtained donor oligonucleotides were used for injection.

(5) The active sgRNA, cas9 mRNA and donor oligonucleotide are mixed and then microinjected into fertilized eggs of SD rats, and then the injected embryo is transplanted into oviduct of a surrogate SD rat for development.

(6) PCR identification of positive Gene-edited rats, marking by toe cut method 9-14 days after birth of F0 rats, and collecting cut tissue samples. Genomic DNA was extracted and PCR detection was performed with specific primers to confirm gene editing, the detection primer sequences were as follows:

5- 'TTCAATGCATTCCTCCTGCT-3' (SEQ ID NO. 3);

The downstream primer is 5- 'TCTTACCCTTGTTGGCCTTG-3' (SEQ ID NO. 4).

PCR identifies positive ones as experimental group and negative ones as littermate wild type control, and F0 rats 5, 7, 19, 31 with target gene point mutation are confirmed again by sequencing PCR products. As shown in FIG. 2, F0 rat was mutated from G to A at nucleotide 38 of the third exon of atcay gene and the corresponding amino acid was changed from glycine to serine.

(7) Mating the obtained F0 positive rats with wild SD rats, breeding to obtain F1 generation rats, and carrying out PCR identification and sequencing to confirm that 12F 1 generation heterozygous rats are obtained, wherein the body of the F1 generation heterozygous rats is free from distortion, the limbs are free from tremors, the F0 positive rats can creep normally, have feeding capacity, and the phenotype of the F1 generation heterozygous rats is free from difference with that of normal rats.

(8) F1 generation heterozygote rats are selfed to obtain F2 generation rats, and 38F 2 generation ATCAY:c.115G > A homozygous mutant rats are obtained through PCR identification and sequencing verification. Phenotype observations were performed on F2 homozygous mutant rats and heterozygous rats, and it was found that F2 homozygous mutant rats had a phenotype (FIG. 3) that developed after one to two weeks of birth, and that the phenotype was significantly different from that of normal rats. The upper and lower bodies of the sick mice are reversely twisted, the limbs of the sick mice have dystonia phenotypes such as high-frequency tremors, and the like, the sick mice can not normally crawl and can not independently eat food, manual feeding is needed three times a day (if manual feeding is not needed, the survival time is not longer than 1 month old), the sick mice accord with the condition of the Kasman ataxia phenotype, the sick mice can be used as an effective model for researching the Kasman ataxia, and the phenotype of the F2 heterozygous mutant rats is not different from that of normal rats.

While the discovery, identification, and example phenotypes of the present invention have been shown and described by way of example, as previously described, it should be understood that the present invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments, but is applicable to various other combinations, modifications, and variations within the scope of the invention as contemplated by the foregoing teachings or by the skilled artisan or knowledge of the relevant arts. Modifications and variations which would be apparent to those skilled in the art are intended to be included within the scope and spirit of the invention.

Claims (4)

1. A construction method of a Kaimachia rat model is characterized by comprising the following steps of mutating 38 th nucleotide of ATCAY gene exon 3 of a rat into A:

(1) Obtaining active sgrnas, cas9 mRNA;

the sequence of the sgRNA is GGATCACCTGGGCGGCACGG-TGG;

(2) Mixing the active sgRNA, cas9 mRNA and a donor oligonucleotide sequence, microinjection into a fertilized egg of a rat, and transplanting the fertilized egg into a oviduct of a surrogate rat for inoculation to obtain an F0 generation rat;

the sequence of the donor oligonucleotide is ACCGCTCCCAGAAGACACCGGGGAGGATCACCTGGGCAGCACGGTGGAAGACTCCTCCT;

The rat is SD rat;

(3) The F0 generation positive rat is hybridized with a wild rat to obtain an F1 generation ATCAY gene heterozygous mutant rat, the F1 generation ATCAY gene heterozygous mutant rat is selfed to obtain an F2 generation ATCAY gene homozygous mutant rat, and the Kasman ataxia rat model is obtained.

2. A targeting system for constructing a Kaman ataxia rat model is characterized by comprising sgRNA, donor oligonucleotides and Cas9 mRNA, wherein the targeting system is used for mutating 38 th nucleotide of ATCAY gene exon 3 of a rat into A;

the sequence of the sgRNA is GGATCACCTGGGCGGCACGG-TGG;

the sequence of the donor oligonucleotide is ACCGCTCCCAGAAGACACCGGGGAGGATCACCTGGGCAGCACGGTGGAAGACTCCTCCT;

the rat is SD rat.

3. Use of a rat model constructed by the method of claim 1 in the study of ataxia disease, wherein the use is for the purpose of non-disease treatment.

4. Use of a rat model constructed by the method of claim 1 for screening or preparing a medicament for preventing or treating kafimbriae ataxia, wherein the use is for the treatment of non-disease.