CN110846330B

CN110846330B - Mutant gene and application thereof in constructing small pig model with webbed foot disease

Info

Publication number: CN110846330B
Application number: CN201810952553.5A
Authority: CN
Inventors: 周琪; 赵建国; 海棠; 贾启涛; 曹春伟; 王红梅; 张颖; 郑千涛; 王霄
Original assignee: Institute of Zoology of CAS
Current assignee: Institute of Zoology of CAS
Priority date: 2018-08-21
Filing date: 2018-08-21
Publication date: 2021-08-27
Anticipated expiration: 2038-08-21
Also published as: CN110846330A

Abstract

The invention provides a miniature pig RIPK4 mutant gene, which has c.1397_1398insA mutation compared with the wild pig RIPK4 gene. The invention provides a construct, which comprises the RIPK4 mutant gene. The invention also provides a recombinant cell obtained by transforming a receptor cell with the construct. The invention provides a method for preparing a small pig model with webbed foot disease, which comprises the following steps: the RIPK4 gene of the miniature pig is changed, so that adenine A is inserted between 1397 th base and 1398 th base of the RIPK4 gene which is positioned in the No. 8 exon. The RIPK4 mutant gene provided by the invention is used for preparing a large-scale webbed-foot disease pig model, the pathogenesis of the webbed-foot disease is researched, and the invention has great guiding significance for clinical prevention, diagnosis and treatment of the webbed-foot disease.

Description

Mutant gene and application thereof in constructing small pig model with webbed foot disease

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to an RIPK4 mutant gene, application of the mutant gene in constructing a small pig model with webbed foot disease (Bartscoas-Papas syndrome) and a construction method of the small pig model with the webbed foot disease.

Background

Miniature pigs are a very good animal model for studying the etiology, molecular mechanisms and treatment of human diseases. The Bama miniature pig is a local pig breed special for China, is mainly produced in Guangxi province of China, and is characterized by typical 'two-head black' appearance, namely, hairs on the head and the tail of the hip are black, and the other parts are white. In recent years, the Bama pigs have the advantages of small size and similar anatomical structure to human, and are gradually considered as good materials for constructing large animal models for human disease medical research by the scientists, so the Bama pigs have extremely high breeding value.

Webbed-foot disease (bartscas-pasas syndrome) is a low-morbidity hereditary birth defect in newborns, often leading to death, mainly manifested as cheilosis/cleft palate symptoms, but also other abnormalities, such as skin folds, merging toes (syndactyly), genital abnormalities, etc.

The existing Bartsocas-Papas syndrome disease model is a mouse, but the physiological structure of the mouse is far away from that of a human, so that the disease model needs to be constructed in a large animal similar to the physiological structure of the human, and support is provided for pathological research, drug screening, pharmacodynamic evaluation and other researches aiming at the disease.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the RIPK4 mutant gene of the miniature pig, the phenotype and the genetic pattern of the transgenic family miniature pig with the mutant gene are consistent with human webbed foot disease (Bartsocas-Papas syndrome), and the mutant gene can be used as a large animal model of the human genetic disease, thereby providing support for pathological research, drug screening, drug effect evaluation and other researches aiming at the disease.

In one aspect, the invention provides a minipig RIPK4 mutant gene having a mutation of c.1397 — 1398insA compared to the wild-type pig RIPK4 gene.

Specifically, the mutation site of the mutant gene is located in the No. 8 exon, and the adenine (A) is inserted between 1397 base and 1398 base, so that the code shift mutation of the coded amino acid occurs (p.Asn466LysfsTer169). Specifically, the 466-position asparagine (Asn) of the RIPK4 mutant gene is mutated into lysine (Lys), and the later translated amino acid is subjected to frame shift mutation.

Preferably, the RIPK4 mutant gene sequence is shown as SEQ ID NO. 29.

Preferably, the miniature pig is a bama miniature pig.

In another aspect, the invention provides a construct comprising the RIPK4 mutant gene.

The invention also provides a recombinant cell obtained by transforming a receptor cell with the construct. Preferably, the recombinant cell is a porcine cell, more preferably a bama miniature porcine cell.

According to the embodiment of the invention, the genome of the recombinant animal cell has a nucleic acid sequence coding a mutant RIPK4, wherein compared with a wild-type RIPK4, the protein of the mutant RIPK4 has mutation of asparagine (Asn) at position 466 in translation to lysine (Lys) and the later translated amino acid has frame shift mutation.

In yet another aspect, the present invention provides a method of preparing a small pig model for webbed foot disease, the method comprising:

the RIPK4 gene of the miniature pig is changed, so that adenine A is inserted between 1397 th base and 1398 th base of the RIPK4 gene which is positioned in the No. 8 exon.

In some embodiments of the invention, according to actual needs, by using genetic engineering technology, the RIPK4 gene of a normal pig is changed, adenine A is inserted between the 1397 th base and the 1398 th base, and a flipper-foot-affected pig model is obtained. In other embodiments, genetic engineering techniques may be used to alter the corresponding sites of RIPK4 gene in animals other than pigs to obtain the desired large or small animal models of webbed foot disease, such as a monkey model of webbed foot disease, a mouse model of webbed foot disease, and the like.

The invention also provides a method for screening the small pig model with the webbed foot disease of RIPK4 gene mutation, which comprises the following steps:

1) extracting nucleic acid DNA of a biological sample to be detected;

2) determining the sequence of the nucleic acid DNA;

3) a sequence of the nucleic acid, or a complement thereof, having a c.1397 — 1398insA mutation compared to the wild-type RIPK4 gene, the mutation being indicative of webbed foot disease;

the biological sample is selected from at least one of blood, skin, hair, and muscle.

In step 2), determining the sequence of the nucleic acid comprises the steps of:

carrying out PCR by using a specific primer of a pig RIPK4 gene to obtain an amplification product and sequencing the amplification product;

preferably, the sequence of the forward primer (F) is shown as SEQ ID NO. 27, and the sequence of the reverse primer (R) is shown as SEQ ID NO. 28.

SEQ ID NO:27 5’-3’:CGCCCCTCTAGGTCTCAGAT；

SEQ ID NO:28 5’-3’:GGCATTGACGCTGATCTTGC

The invention also provides a kit for screening the RIPK4 gene mutation flipper foot disease miniature pig model, which comprises a liquid or powdered specific primer of the pig RIPK4 gene. The kit may include other reagents required for PCR, such as buffers, dntps, polymerase; reagents and consumables required for recovering PCR products, such as a sol solution, a collection tube, a washing solution and the like, can also be included. The DNA of a sample to be detected is used as a template, the kit and the method for screening the RIPK4 gene mutation webbed-foot sick pig are used for detection, the operation is simple and convenient, and a large number of samples can be rapidly identified.

In order to obtain a large animal model with webbed foot disease, ENU (N-ethyl-N-nitrosourea) chemical mutagenesis is carried out on the Bama miniature pig, and ENU is injected into a wild male miniature pig to obtain an F0 generation miniature pig; mating the miniature pig with a wild type female miniature pig of the same breed to obtain an F1 generation miniature pig; mating the F1 generation male miniature pig with a wild type female miniature pig to obtain an F2 generation miniature pig; the F1 generation miniature pig male miniature pig and the F2 generation miniature pig mate to obtain an F3 generation miniature pig (51 wild boars, 34 wild sows, 12 deformed boars and 9 deformed sows, wherein the deformed proportion is 21/106-19.81%), the F3 generation miniature pig is subjected to phenotype screening to obtain a family miniature pig with a webbed-foot disease phenotype, and the family miniature pig carries a RIPK4 homozygous mutant gene.

The webbed podopathy-like inheritance pattern of the family miniature pig conforms to Mendelian inheritance law of autosomal monogenic recessive inheritance (51 normal phenotype boars, 34 normal phenotype sows, 12 malformed boars and 9 malformed sows, wherein the malformation proportion is 21/106-19.81%). The number of wild-type individuals and mutant phenotype individuals in the F3 generation were counted and aligned, since wild-type: the mutant ratio is about 1:1, and the separation law of Mendelian dominant inheritance 1:1 is met; also, the ratio of existing females to males in the mutant was close to 1:1, indicating that the mutant phenotype is independent of sex, thereby determining that the mutant phenotype is autosomal dominant.

In one embodiment according to the present invention, the injected dose of the ENU is 50 to 100mg/kg, more preferably, the injected dose of the ENU is 60 to 70 mg/kg; further preferably, the injection dose of the ENU is 65 mg/kg.

In one embodiment according to the present invention, further comprising: and (3) detecting the semen quality of the wild type male miniature pig after ENU injection, and mating the wild type male miniature pig after injection with the wild type female miniature pig of the same variety after the semen quality of the wild type male miniature pig after injection returns to a normal level.

The phenotypic analysis result shows that the small pig model with the web-foot disease has the advantages of obviously reduced body type, black spots on the back skin, even complete blackening, adhesion of ears on the skin, fusion of mouth and nose, no tail, short limbs and incomplete toe development.

Through whole genome correlation analysis, the pathogenic gene of the small pig model with the webbed foot disease is determined to be RIPK4 gene, and the exon of RIPK4 gene is completely sequenced, so that the mutation site of the mutant gene is positioned between No. 8 exon, 1397 base and 1398 base, adenine A is inserted, the 466 asparagine (Asn) in protein translation of RIPK4 can be mutated into lysine (Lys), and the later translated amino acid has frame shift mutation. According to data retrieval, the RIPK4 gene is well conserved in mammals such as human beings, monkeys, pigs, cattle, sheep, horses, cats, dogs, rabbits, mice, rats and the like, so that the RIPK4 mutant gene provided by the invention is used for preparing a large-scale webfoot disease pig model, researching the pathogenesis of the webfoot disease and has great guiding significance for clinical prevention, diagnosis and treatment of the webfoot disease.

In still another aspect, the present invention provides the use of the genetically mutant animal of the present invention as a model for studying webbed foot disease in humans. Preferably, the genetically mutant animal is a bama minipig.

In one embodiment according to the invention, the invention determines the mutation position by performing genetic linkage analysis and gene clone sequencing on a miniature pig model; preferably, the mutation site closely linked to RIPK4 site is obtained by gene cloning sequencing.

In a further aspect, the present invention provides the use of the mutant gene, construct, recombinant cell or kit of the present invention in the preparation of an animal model for screening for the treatment and/or prevention of webbed foot disease; preferably, the animal model is a mammalian model; more preferably, the mammal is a mouse, monkey or miniature pig.

The phenotype of the miniature pig with the RIPK4 gene mutation is very similar to human webbed foot disease, so that a very good animal model of the human webbed foot disease is provided, and the miniature pig with the RIPK4 gene mutation can be used for pathological research, treatment mode research and drug screening of the human webbed foot disease. In addition, the method provided by the invention avoids hybridization of the Bama pigs and other white pig breeds, does not introduce exogenous genetic information, and thus does not increase the complexity of the genetic background of the Bama pigs, which is beneficial to the establishment of the next purification genetic background and inbred line.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a whole body phenotype of a Bama miniature pig model (Mut) and a wild type piglet (WT) of the present invention;

FIG. 2 is the genotyping results of the Bama minipig model (Mut) and wild type piglets (WT) of the webfoot disease of the present invention, with the mutation sites of the coding regions of the genes indicated by arrows;

FIG. 3 shows the skin of a small pig with a webbed foot disease of the present invention and the skin of a wild type pig are HE-stained, wherein WT is the wild type pig and Mut is the small pig with a webbed foot disease of the present invention, and as a result, the horned layer of the epidermis of the mutant pig is found to be remarkably abnormal, mainly as the horned layer is more loose;

FIG. 4 shows the expression of RIPK4 protein of a small pig model with webbed foot disease and a wild type piglet, the inventors constructed in vitro overexpression vectors RIPK4-HA-WT and RIPK4-HA-Mut and transfected 293T cells, and then detected the expression of RIPK 4; the results show that the protein is significantly degraded after RIPK4 mutation, which indicates that the mutation affects the stability of the protein.

Detailed Description

The present invention is further described below in conjunction with the following figures and examples, it being understood that the examples are intended to further illustrate and explain the present invention, and are not intended to limit the present invention.

The Guangxi Bama miniature pig is purchased from third army medical university and bred in the northern big animal research base of animal research institute of Chinese academy of sciences

ENU Sigma(N8509bulk package)

Anesthetic (ketamine) northeast university of agriculture

Jiangxi Baokeming injection Jiangxi Boda animal medicine health-care company

Example 1 establishment of genetic pedigree of Palma minipig with webbed foot disease

1) ENU is injected into a Guangxi Bama miniature boar, the injection dosage is 65mg/kg, the injection frequency is once per week and is continuous for three weeks,

2) and (3) semen quality detection, wherein the quality of the mutagenized boar semen is detected, the detection indexes comprise boar semen volume, sperm density, sperm survival rate and teraticity rate, and the boar semen is mated with a wild sow after the quality returns to a normal level to generate F1 generation.

3) Mating the miniature boar of the F1 generation with the wild female miniature boar of the same breed to obtain the miniature boar of the F2 generation;

4) mating the F1 generation miniature boar with the F2 female miniature boar of the next generation to obtain an F3 generation miniature boar;

5) specific phenotypes were screened on a large scale and observed in F3 pigs.

6) The F1 boar and the F2 sow are mated to generate an F3 generation, and the inheritance pattern of the F3 generation is determined. And (4) carrying out phenotype screening on the newborn pig to obtain the Guangxi Bama miniature pig with developmental deformity.

7) In the mutant family, 85 individuals with black two ends and 21 individuals with malformation are born together in the F3 generation, and chi fang test (X)²＝1.5220<3.81，P>0.05) it could be determined that the mutation is according to a Mendelian inheritance pattern.

As shown in figure 1, "RIPK 4-/-" is an obtained small pig model of human webbed foot disease, and the phenotype of the pig model is that the limbs are short, the large part of the back is black, the mouth and the nose are fused, and the like; "+/+" is a wild type Guangxi Bama miniature pig with a phenotype of two black heads.

Example 2 genetic location of Gene mutation in the genetic family of Bama miniature pigs with webbed foot disease

Determining mutation positions by carrying out genetic linkage analysis and gene cloning sequencing on F3 generation pigs, extracting DNA of ear tissues of F3 generation pigs, carrying out PCR reaction by designing primers, and carrying out electrophoretic separation on PCR products to obtain mutation sites closely linked with RIPK4 sites.

DNA extraction and quality detection

Firstly, putting a proper amount of ear tissues into a 1.5ml centrifuge tube, cutting the ear tissues into pieces by using small scissors, adding 500 mu L of SNET solution and 10 mu L of proteinase K, and digesting the pieces overnight at 55 ℃ and 200rmp in a shaking table;

② after the tissue is completely digested, centrifuging for 1min at 12000rmp to precipitate hair, transferring the supernatant to a new 1.5ml EP tube;

③ adding 500. mu.L of phenol: chloroform: isoamyl alcohol 25: 24: 1, shaking for 30min at normal temperature;

fourthly, centrifuging the solution at 12000rmp for 15min, carefully sucking 200 mu L of supernatant and transferring the supernatant to a new centrifuge tube to avoid the middle protein layer from swinging;

adding 200 μ L isopropanol into the supernatant, slightly inverting for about 1min, 12000rmp, and centrifuging for 15 min;

sixthly, a small amount of white precipitate can be seen at the bottom of the centrifugal tube, the solution is poured out, the precipitate is not poured out, 1ml of 70 percent ethanol is added, and the bottom of the centrifugal tube is flicked to float the precipitate;

seventhly, 12000rmp is centrifuged for 5min, DNA precipitate is reserved, ethanol is sucked out, and the DNA is dried at room temperature;

adding 100 mu LTE, standing for 10min, and lightly blowing the solution by using a gun to dissolve the DNA;

ninOdrop is used for detecting the concentration and the quality of the DNA solution, the concentration of the DNA is more than 100 ng/mu L, A260/280 is between 1.8 and 2.0, and A260/230 is more than 2;

r to 100 ng/L, 2. mu.L of DNA solution is pipetted and mixed with 1. mu.L of 10 × loadingbuffer, and electrophoresis is performed on 1% agarose gel at 120V for 20 min. The DNA sample with qualified quality has clear main band without fracture, protein and RNA pollution.

2. Primer design

Primer design is carried out on the sequence of the exon of the target gene RIPK4 gene in NCBI database by using Primer5 software, and the designed Primer sequence (Table 1) is sent to Invitrogene for Primer synthesis.

TABLE 1 primer sequences

PCR amplification

PCR using Tiangen 2 XTaq PCRmix, 25. mu.L System

The following PCR amplification procedure was used:

at 94 ℃, pre-denaturation for 3 min; denaturation at 94 ℃ for 30s, annealing at 50-65 ℃ for 30s, and extension at 72 ℃ for 1min for 30 cycles; finally incubation at 72 ℃ for 10 min.

The PCR product was detected by electrophoresis in 1.5% agarose gel electrophoresis and then subjected to the next sequencing. If the amplified sequence is homozygous A insertion (c.1397 — 1398insA), the corresponding pig is a disease-causing pig.

As shown in fig. 2, the lower part is the genotype identification result of the bama miniature pig model of the present invention, and the upper part is the genotype identification result of the wild type guangxi bama miniature pig. Wherein the sequence of the mutant gene of the minipig model of the webfoot disease Bama is shown in SEQ ID NO. 29.

Wherein, bold ATG is the initiation codon; the stop codon is bold in italics.

The mutation site of the mutant gene is underlined and located in the No. 8 exon, and adenine A is inserted between the 1397 base and the 1398 base, so that asparagine (Asn) at position 466 in protein translation of RIPK4 is mutated into lysine (Lys)

SEQ ID NO:29

Example 3 characterization of genetic pedigree of Bama miniature pigs with webbed foot disease

Phenotypic analysis shows that the mutant pig has obviously reduced body type, black spots on the back skin, even totally black skin, ears adhered to the skin, mouth and nose fusion, no tail, shorter limbs and incomplete toe development. The RIPK4 knockout mouse shows skin epidermal differentiation abnormality, and the inventor performs HE staining on the skin of the mutant pig (figure 3), and the result shows that the horny layer of the epidermis of the mutant pig has remarkable abnormality, mainly shows that the horny layer is looser.

Example 4 expression of protein of RIPK4

As no RIPK 4N-terminal antibody can be used in pigs, the inventors constructed in vitro over-expression vectors RIPK4-HA-WT and RIPK4-HA-Mut and transfected 293T cells, and then tested the expression of RIPK 4. The results show that the protein is significantly degraded after RIPK4 mutation, indicating that the mutation affects the stability of the protein (fig. 4).

Example 5 preparation of animal model of genetic engineering disease of Palma minipig with webbed foot disease

Through efficient genetic modification mediated by a CRISPR/Cas9 system, the RIPK4 gene mutation site (c.1397_1398insA) is targeted and introduced into the corresponding sequence of the RIPK4 gene of a normal pig, and the positive pig shows a webbed foot disease phenotype and is obviously inherited.

1. Designing a gRNA, and designing the gRNA capable of targeting sequences near a mutation site (c.1397_1398insA) by using an exon 8 as a template;

2. in-vitro cutting experiments identify the targeting efficiency of the gRNA in the step 1;

3. after transfecting fetal fibroblasts with the Cas9 plasmid and the gRNA plasmid selected in step 2, positive cells were selected and identified. Expanding and freezing the screened cells containing RIPK4 gene mutation sites (c.1397_1398 insA);

4. and 3, taking the fetal fibroblast in the step 3 as a nuclear donor, and performing somatic cell nuclear transfer and embryo transfer to obtain the animal model of genetic engineering diseases of the bama minipigs with the webbed foot disease.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

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cgtggcgccc accgggaggc ggtgaccgcg gagggctgca ccgccctgca cctggcctcc 2100

cgcagcggcc acctggcgac cgtcaagctg ctgctggagg agaaggccga cctgctggcc 2160

cgggggcccc gcagccagac ggcgctgcac ctggccgcgg ctggcggcca ctcggaggtg 2220

gtggaggagc tggtgtgcgc cgacgtgctc gacctatcgg acgagcaggg gctcagcgcg 2280

ctgcacctgg ccgcccaggg ccggcacacc aagaccgtgg agacgctgct cagacacggg 2340

gcccacgtca acctgcagag cctcaagttc cagggtggcc ccggccccgc cgccacgctc 2400

ctgcggcgga gcaagaccta gcctgctgcc ctgggagatg gggcccgcgg ggtccctgtc 2460

ccagcgctgt gctccgctcg ccgtgtgtgg ccgtcgagag gcaggtggct gctgactctg 2520

gattcgcgcc agcatctgct tgggcgtcac ccttgggacg gcgtcggtct gggtgggctc 2580

gcccatccgc ccgtcggtgc accggtgtcg gaggggtggg ggctcggagg catctcgccg 2640

cgcttctggc tcgtgacacg ggccccttgg tgacgaaacc gagagcccac cgtcctgggc 2700

cgagctgccg ccaaccgccc ggaacgcacc gtctggggag gcaggtgtgg gaggagctgt 2760

gtcttttctc tccatttgca gcggcaggcg gagaggcctg ttccagaacg ttgtagcgga 2820

gtgcctgttt catcgtgtgt cgtcttagac gggcgctgtc gactgctgct tagacgcagt 2880

caaaccattc ccgttgttgg gtggtccggt cccggagtga ctgtaacgtg agtggttggt 2940

ctttaagcca caacccatca tcggacctgc tctcttctag ccgcacgcat tcgtccgtcg 3000

gtcagtcctc gtgagcggcc acccacgtgt gctgacccgc ggcctcgtgc atttctcacc 3060

gtagaagaca tcgagcgctg gcgtcaaatg tcggcgtcct gaaggatggg ctgacaacct 3120

cagtttggga ctcagaacag tgttccctgc ttagagggtc ccacctttct cctcattttc 3180

tttccagatc tcgtttttgg caaagggggg tggtttgtgc tgagtgatgc tgttttgggg 3240

tccccccggg ggtttctgac ctgctttgca aacagctaca ctgtgcaggg gcctcggctt 3300

tgggggggtg acccccgagt cgggtccaga agacagcctc atggtctcgg acgtagaagc 3360

cccgttcttt cattgtcacc tgtgtcgcac atgccactgt tggggtgagt tggccgcagg 3420

ccctcccctg tgtgcggaca tggccgtcgg ccgggcgtgt gagtcagaga tcgatgctga 3480

ttgatgtacc gtatgtgttc atatgattct gtggacagga ccgttctttt ctatgacagg 3540

aaatatccag actgcttaga actggctatg ttttaatagg cctcgtgctt ttaatatgtt 3600

accctatggt gatatgccga acacgtggaa gaaaaaggtt ttctttgata tcaataaagc 3660

tgttttcttc ctcctccttc aaa 3683

Claims

1. Of miniature pigsRIPK4Mutant gene, said mutant gene and wild type pigRIPK4A mutation of c.1397 — 1398insA in gene comparison;

wherein, theRIPK4The sequence of the mutant gene is shown in SEQ ID NO. 29.

2. A construct comprising the construct of claim 1RIPK4MutationsA gene.

3. A recombinant cell obtained by transforming a recipient cell with the construct of claim 2.

4. The recombinant cell of claim 3, wherein the recombinant cell is a porcine cell.

5. The recombinant cell of claim 3, wherein the recombinant cell is a Bama miniature pig cell.

6. A method of making a mini pig model of webbed foot disease, the method comprising:

for changing pigletsRIPK4Gene ofRIPK4Adenine A is inserted between 1397 th base and 1398 th base of the gene,

wherein, after modificationRIPK4The sequence of the gene is shown in SEQ ID NO. 29.

7. A screening method for non-diagnostic purposes hasRIPK4A method of mutating a minipig model of webbed foot disease of a gene, the method comprising the steps of:

1) extracting nucleic acid DNA of a biological sample to be detected;

2) determining the sequence of the nucleic acid DNA;

3) the sequence of the nucleic acid or the complementary sequence thereof, with the wild typeRIPK4The gene has a c.1397 — 1398insA mutation compared to the gene, which mutation is indicative of webbed foot disease;

wherein the mutant formsRIPK4The sequence of the gene is shown as SEQ ID NO. 29, and the biological sample is at least one selected from blood, skin, hair and muscle.

8. The method according to claim 7, wherein in step 2), determining the sequence of the nucleic acid comprises the steps of:

by pigsRIPK4PCR is carried out on the specific primer of the gene to obtain an amplification product and the amplification productSequencing the substance;

wherein, the sequence of the forward primer is shown as SEQ ID NO. 27, and the sequence of the reverse primer is shown as SEQ ID NO. 28.

9. Screening methodRIPK4The kit of the gene mutation webbed foot disease miniature pig model is characterized by comprisingRIPK4Specific primers for the gene;

10. Use of the mutant gene of claim 1, the construct of claim 2, the recombinant cell of any one of claims 3 to 5, or the kit of claim 9 in the preparation of an animal model for screening for webfoot disease.

11. The use of claim 10, wherein the animal model is a mammalian model.

12. The use of claim 11, wherein the mammal is a mouse, monkey, or mini pig.