CN117143916A - Method for establishing WNT10A gene function-deficiency mutation disease model dog - Google Patents

Abstract

The invention relates to a method for establishing a WNT10A gene deletion function variation disease model dog, and the obtained WNT10A gene knockout type model dog, cells, tissues and organs thereof, wherein the model dog presents permanent tooth development disorder which belongs to primary symptoms, has long duration of clinical phenotype, strong stability and inheritability.

Description

Method for establishing WNT10A gene function-deficiency mutation disease model dog

Technical Field

The invention belongs to the field of genetic engineering, and in particular relates to a method for establishing a WNT10A gene function deletion mutation disease model dog by using a gene editing technology.

Background

Teeth are important organs of the oromaxillofacial region, and the normal number of teeth is the basis for performing physiological functions. Congenital tooth loss (hereinafter referred to as congenital tooth loss) is one of the common genetic diseases, and affects the chewing, pronunciation, beauty of patients, etc.

Mutations in various genes were found to be associated with congenital dental defects by clinical genetic analysis in human congenital dental defect patients. Among the common pathogenic genetic variations is the WNT10A (winter-type MMTV integration site family, member 10A) gene. In non-syndromic majority of tooth-deficient patients (more than 6 teeth were congenital missing), the carrier frequency of WNT10A mutations was as high as 56%. The WNT10A gene mutation can cause congenital tooth deficiency in an autosomal dominant and autosomal recessive genetic mode, and the functional influence of the gene mutation and the tooth deficiency performance show a certain dose dependency relationship.

The gene knockout mouse model is an experimental animal model which is more commonly used for researching tooth development. However, WNT10A knockout mice did not exhibit congenital tooth-deficient performance similar to human WNT10A mutant patients. Since the traditional tooth development research mostly uses rodents as a research model, the related research of human milk permanent tooth replacement cannot be performed without milk permanent tooth replacement. If the influence of the double allele functional deletion variation of WNT10A on the development of permanent teeth is to be studied, the gene knockout mouse model cannot be used.

Therefore, it is necessary to artificially create a WNT10A gene-edited canine that provides a superior animal model for the study of WNT10A gene-dysfunction disease.

Disclosure of Invention

The invention provides a method for establishing a WNT10A gene deletion function variation disease model dog by a gene editing technology. The invention also relates to the established WNT10A gene deletion function variation disease model dogs, cells and tissues thereof.

One aspect of the present invention provides a method for constructing a model canine for a WNT10A gene deletion functional variation disease, the method comprising obtaining a canine fertilized egg or canine somatic cell with reduced or deleted WNT10A gene expression using a gene editing technique.

In some embodiments, the gene editing technique is selected from BE3 single base editing techniques, CRISPR, TALEN, and ZFN, preferably CRISPR/Cas9.

In some embodiments, the method comprises targeted mutation of exon 2 of the WNT10A gene, preferably the mutation comprises an insertion, substitution, or deletion of a nucleotide.

In some embodiments, the method comprises the steps of:

(1) Determining a target site according to the sequence of exon 2 of the canine WNT10A gene;

(2) Synthesizing an sgRNA sequence according to the targeting site determined in the step (1), and then connecting the synthesized sequence with a skeleton carrier to construct an sgRNA targeting carrier;

(3) In vitro transcription products of sgRNA and CRISPR/Cas9 are obtained by in vitro transcription, respectively;

(4) And (3) introducing the sgRNA and the CRISPR/Cas9 in vitro transcription product obtained in the step (3) into a canine fertilized egg or canine somatic cell to obtain the canine fertilized egg or canine somatic cell with reduced or deleted WNT10A gene expression.

In some embodiments, 3 sgRNAs are determined from the sequence of exon 2 of the canine WNT10A gene,

preferably, the sequence of the sgRNA and its complement comprises the following sequences:

sgRNA1：GCGGAGGCCCAGAATGTCGTTGG(SEQ ID NO:2)

complementary sequence of sgRNA 1: CCAACGACATTCTGGGCCTCCGC (SEQ ID NO: 3)

sgRNA2：CAACGTTAGGCACACCGTGTTGG(SEQ ID NO:4)

Complementary sequence of sgRNA 2: CCAACACGGTGTGCCTAACGTTG (SEQ ID NO: 5)

sgRNA3：GGCACTCATGGATGGCGATCTGG(SEQ ID NO:6)

Complementary sequence of sgRNA 3: CCAGATCGCCATCCATGAGTGCC (SEQ ID NO: 7).

In some embodiments, the method further comprises transplanting the canine fertilized egg with reduced or deleted WNT10A gene expression into the oviduct of a recipient female canine, thereby preparing a model canine for a WNT10A gene deleted functional variation disease.

In other embodiments, the method further comprises transplanting the nucleus of the canine somatic cell with reduced or deleted WNT10A gene expression into a canine enucleated oocyte, and then transplanting the nucleus-transplanted canine enucleated oocyte into the oviduct of a recipient female canine, thereby preparing a model canine for the WNT10A gene-deleted functional variant disease.

In some embodiments, the canine somatic cells are from a tissue or organ selected from the group consisting of: fetal tissue, skin, muscle, ear, breast, fallopian tube, ovary, blood, urine, fat, bone marrow, blood vessels, and luminal endothelium.

In some embodiments, the canine somatic cell is selected from the group consisting of fetal fibroblasts, skin cells, epithelial cells, ear cells, fibroblasts, endothelial cells, muscle cells, breast cells, oviduct cells, ovarian cells, cumulus cells, nerve cells, and osteoblasts.

In some embodiments, the genome of the WNT10A gene deletion functional variant disease model canine comprises a nucleotide sequence as set forth in at least one of SEQ ID NOs 10, 12, 14, 16, and 18.

In some embodiments, the model canine for a disease with a deletion function of the WNT10A gene expresses a protein having an amino acid sequence as set forth in at least one of SEQ ID NOs:11, 13, 14, 15, and 19.

In some embodiments, the backbone vector used may include, in addition to eukaryotic vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, non-viral vectors, and the like.

In some embodiments, the invention utilizes gene editing technology, selects a targeting site sequence according to exons of a canine WNT10A gene sequence, constructs a sgRNA targeting vector and a CRISPR/Cas9 expression vector according to the targeting site sequence, transcribes the vector into mRNA in vitro after verification and is effective, then adopts a cytoplasmic injection mode to inject the mRNA into canine fertilized eggs, and then transplants the canine fertilized eggs into one oviduct of a female canine with both oviducts subjected to embryo punching, thereby preparing the model canine for the WNT10A gene deletion function variation disease.

In another aspect, the present invention provides canine somatic cells, tissues or organs of a canine, which is a model of a disease in which the WNT10A gene is deleted from function, obtained by the method of establishment.

In some embodiments, the canine somatic cells, tissues or organs of the WNT10A gene-deleted functional mutant disease model canine comprise a nucleotide sequence as set forth in at least one of SEQ ID NOs 10, 12, 14, 16 and 18.

In some embodiments, the canine somatic cells, tissues or organs of the WNT10A gene-deleted functional mutant disease model canine express a protein having an amino acid sequence as set forth in at least one of SEQ ID NOs 11, 13, 14, 15 and 19.

In another aspect, the invention also provides canine somatic cells of a model canine for WNT10A gene-deleted functional variation disease, the genome of which comprises a nucleotide sequence set forth in at least one of SEQ ID NOs 10, 12, 14, 16 and 18.

In yet another aspect, the invention provides a targeting vector for editing a canine WNT10A gene, the targeting vector comprising an sgRNA sequence designed for a targeting site sequence determined for exon 2 of the canine WNT10A gene, and a backbone vector;

preferably, the sgrnas and their complements include the following sequences:

sgRNA1：GCGGAGGCCCAGAATGTCGTTGG(SEQ ID NO:2)

complementary sequence of sgRNA 1: CCAACGACATTCTGGGCCTCCGC (SEQ ID NO: 3)

sgRNA2：CAACGTTAGGCACACCGTGTTGG(SEQ ID NO:4)

Complementary sequence of sgRNA 2: CCAACACGGTGTGCCTAACGTTG (SEQ ID NO: 5)

sgRNA3：GGCACTCATGGATGGCGATCTGG(SEQ ID NO:6)

Complementary sequence of sgRNA 3: 5' -CCAGATCGCCATCCATGAGTGCC (SEQ ID NO: 7).

In yet another aspect, the invention provides a cell comprising the targeting vector.

In some embodiments, the cell is unable to develop into an animal.

In yet another aspect of the present invention, there is provided a primer pair comprising the sequence:

forward primer: GTAACCCTTCCCCCAGATGC (SEQ ID NO: 8),

reverse primer: CTCCCTCCCATTTCCGACAC (SEQ ID NO: 9).

In yet another aspect, the invention provides the use of the primer pair in detecting a model canine for a WNT10A gene deletion function variation disease comprising a genomic sequence of at least one of the nucleotide sequences set forth in SEQ ID NOs 10, 12, 14, 16 and 18.

The invention also provides an application of the WNT10A gene deletion function variation disease model dog obtained by the method in screening medicines for preventing or treating permanent tooth development disorder diseases.

A common causative genetic variation in the diseases of permanent tooth development disorder is the WNT10A (Window-type MMTV integration site family, member 10A) gene. In non-syndromic majority of tooth-deficient patients (more than 6 teeth were congenital missing), the carrier frequency of WNT10A mutations was as high as 56%. The WNT10A gene mutation can cause congenital tooth deficiency in an autosomal dominant and autosomal recessive genetic mode, and the functional influence of the gene mutation and the tooth deficiency performance show a certain dose dependency relationship. When only one chromosome of WNT10A had a minor loss of function mutation, the patient had only a few teeth with congenital deletions, accompanied by conical teeth in the maxillary side incisors. Patients typically develop a majority of dental congenital defects when only one chromosome of WNT10A has a more pronounced nonfunctional mutation, or both chromosomes of WNT10A have a more mild nonfunctional mutation. When the WNT10A of both chromosomes of the patient has more pronounced loss-of-function mutations, it is generally manifested as a syndrome, such asSyndrome [ ]syndrome, SSPS) and odontopathy-cutaneous dysplasia syndrome (OODD), with most, even all, permanent congenital deletions, accompanied by a development deficient phenotype of other organs.

In the prior art, rodents are used as research models in the tooth development research, and the related research of human permanent teeth cannot be performed without replacing the milk permanent teeth. Dogs, like humans, have a tooth replacement period and are ideal animal models of diseases related to permanent teeth. The model dog of WNT10A gene deletion function variation disease established by the gene editing technology shows symptoms of the pathogenic diseases of the permanent tooth development disorder, has long duration of clinical phenotype, strong stability and heritability, and can well simulate the clinical symptoms of human beings. In addition, the WNT10A gene deletion function variation disease model dogs obtained by the method can obtain a large number of offspring disease model dogs in a natural breeding mode, and the problem of the shortage of congenital amaurosis animal disease model numbers is also solved. The development of the WNT10A gene editing dog model provides a better theoretical research animal model for human permanent tooth development disorder diseases and provides technical support for the research of pathogenesis of the diseases, the development of treatment means and other technologies.

Drawings

Fig. 1 shows the PCR identification results of puppies numbered 220601 ～ 220605.

Fig. 2 shows the PCR identification results for puppies numbered 220714 ～ 220721.

FIG. 3 shows the results of nucleic acid sequence alignment analysis for puppies numbered 220604.

FIG. 4 shows the results of nucleic acid sequence alignment analysis for puppies numbered 220716.

FIG. 5 shows the maxillary and mandibular milks and dentition intentions of WNT10A gene wild dogs.

Figure 6 shows the tooth control results for 5 month old teeth of dogs numbered 220604 versus wild type dogs.

Figure 7 shows a CT image of a 6 month old tooth of dog numbered 220716.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.

Example 1

1 construction and identification of targeting vectors

The targeting site (SEQ ID NO: 1) was determined from exon 2 of the WNT10A Gene (Gene ID: 488528) in Genbank based on the canine WNT10A Gene sequence information.

3 sgrnas were designed for this targeting site, the sgRNA sequences are shown in table 1 below.

TABLE 1

The vector plasmid px330 was digested and linearized, 1% agarose gel electrophoresed, then cut and recovered, and the concentration was determined and ligated overnight at 16℃using T4 DNA ligase according to a system of linearization vector to WNT10A gene targeting sgRNA molar ratio of 1:3. The ligation product was transformed into ampicillin resistant LB plates, positive clones were identified by colony PCR, and plasmids were extracted by shaking and plasmid miniprep kit. 10 mu L of recombinant plasmid is sucked up and is delivered to sequencing, and the result of sequencing is analyzed and compared by adopting Snapgene, and the plasmid with correct sequencing and comparison is stored for standby.

2 in vitro transcription

Firstly linearizing a CRISPR/Cas9 plasmid, wherein the reaction system is as follows: 30. Mu.g of plasmid, 5. Mu.L of restriction enzyme AflII;10 μL of 10 XBuffer and ddH ₂ O, total volume was 100. Mu.L. Then 100 μl of phenol was added: imitation: purification of linearized plasmid DNA by isoamyl alcohol (25:24:1), 12000g of ionHeart for 5min; sucking 50 μl of the supernatant into a 1.5ml centrifuge tube without RNase, adding 1/10 volume of sodium acetate and 3 times volume of absolute ethanol to precipitate plasmid DNA, centrifuging 12000g for 5min; discarding the supernatant, sucking the residual supernatant as much as possible, adding 150 mu L of 70% ethanol to wash the plasmid, and centrifuging for 5min at 12000 g; air drying for 3-5min, and adding 15 μl of RNase-free ddH ₂ O dissolves DNA and the concentration is determined.

In vitro transcription was performed using an in vitro transcription mRNA kit (Thermo Scientific).

The in vitro transcription system is as follows: 1. Mu.g of linearized plasmid DNA, 10. Mu.L of 2 XNTP/CAP, 2. Mu.L of 10 XBuffer, 2. Mu.L of RNA synthetase and ddH2O in a total volume of 20. Mu.L. Mixing, and incubating at 37deg.C for 1hr; mu.L TURBO DNase was added and the plasmid template digested and incubated at 37℃for 30min. Then, 20. Mu.L of the in vitro transcription product, 20. Mu.L of 10 XReation Buffer, 10. Mu.L of ATP (10 mM), 2.5. Mu.L of RNase inhibitor, 2. Mu.L of Poly (A) polymerase and ddH2O were mixed to prepare an in vitro transcription mRNA-plus-polyA system in a total volume of 100. Mu.L, and incubated at 37℃for 1hr. After incubation, 350 mu L of binding buffer solution is added into the reaction system, and the mixture is blown and sucked uniformly; then 250 mu L of absolute ethyl alcohol is added and mixed uniformly; transferring the sample into an mRNA purification column, and centrifuging 10000g at room temperature for 1min; discarding the filtrate, reloading the column, rinsing the column with 500. Mu.L of eluent, centrifuging 10000g at room temperature for 1min; repeatedly rinsing for one time, discarding filtrate, centrifuging for 1min by using a hollow column, and eluting impurities such as protein; then placing the column into a new centrifuge tube, adding 50 mu L of RNA eluent to the central position of the column, covering a cover, incubating for 10min at 65 ℃, and centrifuging for 1min at 10000g at room temperature; and detecting the quality and concentration of RNA.

The CRISPR sgRNA and Cas9 mRNA were mixed to a final concentration of 20 ng/. Mu. L, cas9 for 200 ng/. Mu.L, and stored at-80℃for cytoplasmic injection.

3 cytoplasmic injection and embryo transfer and identification

Cas9 mRNA and 3 sgRNAs were mixed in a ratio of 2:1, and the fertilized egg cytoplasm of dogs was injected, and embryo transfer was performed 10 times in total, the number of transferred embryos was 52, the number of transfer recipients was 10, and the total number of pups at birth was 20, and 1 positive dog was gene-edited (see Table 2 for details).

The specific operation comprises the following steps: in total, 10 beagle dogs with natural oestrus were used as fertilized egg donors and embryo transfer recipients. And (3) collecting blood of all the female dogs, detecting the concentration of progesterone in serum, determining an ovulation period when the concentration of progesterone reaches 4-7ng/mL, naturally mating 48 hours after ovulation, and then flushing fertilized embryos, wherein 10 female dogs cumulatively obtain 52 fertilized eggs. After fertilized eggs were collected, cumulus granulosa cells were removed using TCM199 medium containing 0.1% hyaluronidase, and then placed in HEPES buffered microdroplets of TCM199 medium (HM, GIBCO 11150) and placed on an inverted microscope equipped with a micromanipulator. The mixed solution containing the mRNA of the sgRNA prepared above and the mRNA of Cas9 in a volume ratio of 4:1 was aspirated with a microinjection needle, and then injected into the cytoplasm of the fertilized egg. The oviduct was rinsed with 10mL of HEPES buffered TCM199 medium (HM, GIBCO 11150) containing 10% fetal bovine serum, and the eggs were removed from the needle ligated by the umbrella of the oviduct and collected in a 10mL centrifuge tube. After the cytoplasmic injection is completed, the embryo is put into an embryo transfer tube, and the embryo in the embryo transfer tube is injected into the oviduct on the side with less bleeding when the embryo is flushed from the umbrella part. A total of 20 puppies were finally obtained.

TABLE 2 cytoplasmic injection and embryo transfer results

After birth of puppies, ear tissue (E) and tail tissue (T) were collected and genomic DNA was extracted using DAN extraction kit (ZhangGO-BTCD-400) for identification. After the tissue blocks are sheared in the centrifuge tube, proteinase K water bath is added for cracking for 1-3h at 56 ℃. Then, genomic Lysis Buffer. Mu.L was pipetted with a pipette, the lysis system was added, mixed well upside down, 10000g was centrifuged for 1min. The supernatant was pipetted into a purification column, 10000g, allowed to stand at room temperature for 1min, and centrifuged for 1min. The collection tube was replaced with a new one, 200. Mu.L of DNA Pre-Wash Buffer,10000g was added to the column, and the mixture was allowed to stand at room temperature for 1min, centrifuged for 1min, and the waste liquid was discarded. 400. Mu.L of g-DNA Wash Buffer,10000g, was added to the column, left to stand at room temperature for 1min, centrifuged for 1min, and the waste liquid was discarded. The purification column and collection tube were re-centrifuged at 10000g for 2min. The purification column was replaced with a new 1.5mL centrifuge tube, 50. Mu.L of the solution Buffer was added to elute the DNA, and the column was left at room temperature for 2min.12000rpm, and centrifuging for 1min, wherein the obtained solution is canine genomic DNA.

PCR and PCR product sequencing identification are carried out by taking canine genomic DNA as a template, wherein a genotype identification primer pair is as follows:

WNT10A-DOG-U1 (forward primer): GTAACCCTTCCCCCAGATGC (SEQ ID NO: 8)

WNT10A-DOG-D1 (reverse primer): CTCCCTCCCATTTCCGACAC (SEQ ID NO: 9)

PCR reaction System (30. Mu.L): forward primer 1. Mu.L and reverse primer 1. Mu. L, KODone enzyme 15. Mu. L, ddH ₂ O12. Mu.L, template 1. Mu.L.

The PCR reaction conditions were: 3min at 98 ℃ (98 ℃ 10s,60 ℃ 10s,68 ℃ 10 s) 30 cycles; 68 ℃ for 5min; preserving at 4 ℃.

The PCR products were cut, purified and recovered, and then connected to T vectors for transformation into E.coli, and 15 colonies were picked per plate for sequencing. And comparing the sequencing result with a wild type sequence, and analyzing the mutation condition of the monoclonal gene to obtain WNT10A gene editing canine individuals with numbers 220604 and 220716.

FIGS. 1 and 2 show PCR identification results for newborn puppies, wherein each band of the gel chart in FIG. 1 represents a DL2000marker, a puppy numbered 220601 ～ 220605 (ear, tail tissue mixed sample), and a negative control (sample is water), respectively, and the amplification results show that the amplification product (arrow) of the puppy sample numbered 220604 is smaller than that of the other puppy samples; each band of the gel diagram in fig. 2 represents DL2000marker, puppy numbered 220714 ～ 220718 (two samples of ear tissue and tail tissue, respectively), negative control (sample water), puppy numbered 220719 ～ 220721 (two samples of ear tissue and tail tissue, respectively), negative control (sample water), and the amplification result showed that the amplification product (indicated by the arrow) of the sample numbered 220716 puppy was smaller than that of the other puppy samples.

Through further sequencing and sequence information comparison, 2 puppies (with numbers of 220604 and 220716 respectively) are found to mutate at the target site of exon 2 of the WNT10A gene, and the gene sequencing results are shown in fig. 3 and 4, and the results show that: the genotype of the WNT10A gene of the canine individual with the code 220604 has 2 genotypes which are 3bp deletion genotype and 51bp deletion genotype; the genotype of the WNT10A gene of the canine individual with the code 220716 has a 105bp deletion genotype, a 103bp deletion genotype, a 51bp deletion, a 1bp insertion and a total of 3 genotypes of the 12bp deletion genotypes. Specific mutation information is shown in table 3 below:

TABLE 3 Table 3

Description of the invention	Sequence number
		220604 nucleic acid sequence of canine-3 bp genotype	SEQ ID NO:10
220604 amino acid sequence of canine-3 bp genotype	SEQ ID NO:11
		220604 nucleic acid sequence of canine-51 bp genotype	SEQ ID NO:12
220604 amino acid sequence of canine-51 bp genotype	SEQ ID NO:13
		220716 nucleic acid sequence of dog-105 bp genotype	SEQ ID NO:14
220716 amino acid sequence of dog-105 bp genotype	SEQ ID NO:15
		220716 nucleic acid sequence of canine-103 bp genotype	SEQ ID NO:16
220716 amino acid sequence of canine-103 bp genotype	SEQ ID NO:17
		220716 nucleic acid sequence of canine-51bp+1bp, -12bp genotype	SEQ ID NO:18
220716 canine-51bp+1bp, -12bp genotype amino acid sequence	SEQ ID NO:19
		Wild type nucleic acid sequence of WNT10A gene No. 2 exon	SEQ ID NO:20
Wild type amino acid sequence of WNT10A gene exon 2	SEQ ID NO:21

220604 sequencing results (-3 bp/-51 bp):

the 3bp deleted nucleic acid sequence is (SEQ ID NO: 10):

aggacaggagaatgctttaggaatatacctataggcccaacctttagaagcaaagattggaacagatggattatggcctttgggacagagtgtaagttgctaaatgggggccctcctagctcccctaaaacactcccccacacctactcccccactgccttcaggtctgcacccaacattctgggcctccgcctccccccggagcctgtgctcaatgccaacacggtgtgcctaacgttgccgggcctgagcaggaggcagatggaagtgtgtgtgcgccacccagacgtggctgcctcagccatccagggcatccagatcgccatccatgagtgccagcaccagttccgggaccagcgctggaactgctccagtctggagactcgcaacaagatcccctacgagagtcccatcttcagcagaggtagctgccccctgccttacccttctgcccacccctccagcacacctccacccccaaagccagccattcccaacccactccctcaagctggcaacctcctcaggtgcccatctaccagccgcgctactcttttctggtcttggaca

the 3bp deleted amino acid sequence was (416 aa, ASP deleted) (SEQ ID NO: 11):

MGSTHPCPRLRLRPRPQPRPALCALLFFLLLLAAAVPRSAPNILGLRLPPEPVLNANTVCLTLPGLSRRQMEVCVRHPDVAASAIQGIQIAIHECQHQFRDQRWNCSSLETRNKIPYESPIFSRGFRESAFAYAIAAAGVVHAVSNACALGKLKACGCDASRRGDEEAFRRKLHRLQLDALQRGKGLSHGVPEHPALPPASPGLQDSWEWGGCSPDVGFGERFSKDFLDSREPHRDIHARMRLHNNRVGRQAVMENMRRKCKCHGTSGSCQLKTCWQVTPEFRAVGALLRSRFHRATLIRPHNRNGGQLEPGPAGAPSPAPGAPGPRRRASPADLVYFEKSPDFCEREPRLDSAGTVGRLCNKSSAGPDGCGSMCCGRGHNILRQTRSERCHCRFHWCCFVVCEECRITEWVSVCK*

-51bp deletion nucleic acid sequence (SEQ ID NO: 12):

gtagctgcctataggggcgaattgggccctctagatgcatgctcgagcggccgccagtgtgatggatatctgcagaattgccctttgtccaagaccagaaaagagtagcgcggctggtagatgggcacctgaggaggttgccagcttgagggagtgggttgggaatggctggctttgggggtggaggtgtgctggaggggtgggcagaagggtaaggcagggggcagctacctctgctgaagatgggactctcgtaggggatcttgttgcgagtctccagactggagcagttccagcgctggtcccggaactggtgctggcactcatggatggcgatctggatgccctggatggctgaggcagccacgtctgggtggcgcacacacacttccatctgcctcctgctcaggcccggcaacgttaggcacacgttgggtgcagacctgaaggcagtgggggagtaggtgtgggggagtgttttaggggagctaggagggcccccatttagcaacttacactctgtcccaaaggccataatccatctgtttcaatctttgcctctaaaggttgggcctataggtatattcctaaagcattctcctgtcctaagggcaattccagcacactggcggccgttactagtggatccgagctcggtaccaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttaatcccgctca

the 51bp deletion amino acid sequence is (code 239 aa) (SEQ ID NO: 13):

VAAYRGELGPLDACSSGRQCDGYLQNCPLSKTRKE*RGW*MGT*GGCQLEGVGWE WLALGVEVCWRGGQKGKAGGSYLC*RWDSRRGSCCESPDWSSSSAGPGTGAGTHGWRS GCPGWLRQPRLGGAHTLPSASCSGPATLGTRWVQT*RQWGSRCGGVF*GS*EGPHLATYT LSQRP*SICFNLCL*RLGL*VYS*SILLS*GQFQHTGGRY*WIRARYQAWRNHGHSCFLCEIV NPA

220716 sequencing results (-103 bp, -51+1, -12bp, -105 bp):

the 105bp deleted nucleic acid sequence is (SEQ ID NO: 14):

aggacaggagaatgctttaggaatatacctataggcccaacctttagaagcaaagattggaacagatggattatggcctttgggacagagtgtaagttgctaaatgggggccctcctagctcccctaaaacactcccccacacctactcccccactgccttcaggtctgcacccaacgacattctgggcctccgcctccccccggagcctgtgctcaacgccatccatgagtgccagcaccagttccgggaccagcgctggaactgctccagtctggagactcgcaacaagatcccctacgagagtcccatcttcagcagaggtagctgccccctgccttacccttctgcccacccctccagcacacctccacccccaaagccagccattcccaacccactccctcaagctggcaacctcctcaggtgcccatctaccagccgcgctactcttttctggtcttggaca

the 105bp deletion amino acid sequence is (coding 382 aa) (SEQ ID NO: 15):

MGSTHPCPRLRLRPRPQPRPALCALLFFLLLLAAAVPRSAPNDILGLRLPPEPVLNAIHECQHQFRDQRWNCSSLETRNKIPYESPIFSRGFRESAFAYAIAAAGVVHAVSNACALGKLKACGCDASRRGDEEAFRRKLHRLQLDALQRGKGLSHGVPEHPALPPASPGLQDSWEWGGCSPDVGFGERFSKDFLDSREPHRDIHARMRLHNNRVGRQAVMENMRRKCKCHGTSGSCQLKTCWQVTPEFRAVGALLRSRFHRATLIRPHNRNGGQLEPGPAGAPSPAPGAPGPRRRASPADLVYFEKSPDFCEREPRLDSAGTVGRLCNKSSAGPDGCGSMCCGRGHNILRQTRSERCHCRFHWCCFVVCEECRITEWVSVCK*

the 103bp deleted nucleic acid sequence is (SEQ ID NO: 16):

aggacaggagaatgctttaggaatatacctataggcccaacctttagaagcaaagattggaacagatggattatggcctttgggacagagtgtaagttgctaaatgggggccctcctagctcccctaaaacactcccccacacctactcccccactgccttcaggtctgcacccaacgacattctgggcctccgcctccccccggagcctgtgctcaatgccaacaccatgagtgccagcaccagttccgggaccagcgctggaactgctccagtctggagactcgcaacaagatcccctacgagagtcccatcttcagcagaggtagctgccccctgccttacccttctgcccacccctccagcacacctccacccccaaagccagccattcccaacccactccctcaagctggcaacctcctcaggtgcccatctaccagccgcgctactcttttctggtcttggaca

the 103bp deletion amino acid sequence is (code 119 aa) (SEQ ID NO: 17):

MGSTHPCPRLRLRPRPQPRPALCALLFFLLLLAAAVPRSAPNDILGLRLPPEPVLNANT MSASTSSGTSAGTAPVWRLATRSPTRVPSSAEVFARAPSPMPSRQLGSCTLCPMPVPWAN*

-51+1, -12 deletion nucleic acid sequence (SEQ ID NO: 18):

aggacaggagaatgctttaggaatatacctataggcccaacctttagaagcaaagattggaacagatggattatggcctttgggacagagtgtaagttgctaaatgggggccctcctagctcccctaaaacactcccccacacctactcccccactgccttcaggtctgcacccaacgTtgtgcctaacgttgccgggcctgagcaggaggcagatggaagtgtgtgtgcgccacccagacgtggctgcctcagccatccagggcatccatgagtgccagcaccagttccgggaccagcgctggaactgctccagtctggagactcgcaacaagatcccctacgagagtcccatcttcagcagaggtagctgccccctgccttacccttctgcccacccctccagcacacctccacccccaaagccagccattcccaacccactccctcaagctggcaacctcctcaggtgcccatctaccagccgcgctactcttttctggtcttggaca

-51+1, -12 deleted amino acid sequence (coding 73 aa) (SEQ ID NO: 19):

MGSTHPCPRLRLRPRPQPRPALCALLFFLLLLAAAVPRSAPNVVPNVAGPEQEADGSV CAPPRRGCLSHPGHP*

wild-type nucleic acid sequence of exon 2 of WNT10A gene (SEQ ID NO: 20)

aggacaggagaatgctttaggaatatacctataggcccaacctttagaagcaaagattggaacagatggattatggcctttgggacagagtgtaagttgctaaatgggggccctcctagctcccctaaaacactcccccacacctactcccccactgccttcaggtctgcacccaacgacattctgggcctccgcctccccccggagcctgtgctcaatgccaacacggtgtgcctaacgttgccgggcctgagcaggaggcagatggaagtgtgtgtgcgccacccagacgtggctgcctcagccatccagggcatccagatcgccatccatgagtgccagcaccagttccgggaccagcgctggaactgctccagtctggagactcgcaacaagatcccctacgagagtcccatcttcagcagaggtagctgccccctgccttacccttctgcccacccctccagcacacctccacccccaaagccagccattcccaacccactccctcaagctggcaacctcctcaggtgcccatctaccagccgcgctactcttttctggtcttggaca

Wild-type amino acid sequence of exon 2 of WNT10A gene (SEQ ID NO: 21)

RTGECFRNIPIGPTFRSKDWNRWIMAFGTECKLLNGGPPSSPKTLPHTYSPTAFRSAPNDILGLRLPPEPVLNANTVCLTLPGLSRRQMEVCVRHPDVAASAIQGIQIAIHECQHQFRDQRWNCSSLETRNKIPYESPIFSRGSCPLPYPSAHPSSTPPPPKPAIPNPLPQAGNLLRCPSTSRATLFWSWT

Example 4 phenotypic analysis of WNT10A Gene-edited dogs

Phenotypic analysis was performed on the WNT10A gene-edited dogs obtained above. Compared with the arrangement of the upper and lower jaw milk and the permanent teeth of a WNT10A gene wild type dog (see figure 5), the first to third permanent premolars of the upper jaw of the No. 220604 dog at 5 months of age are missing (see figure 6), which shows that the No. 220604 dog shows a clinically typical permanent tooth development disorder disease phenotype, and shows that the WNT10A gene function missing variant disease model dog is successfully constructed. CT results (see FIG. 7) of dog No. 220716, 6 months old, show that the first to third permanent premolars of the upper jaw and the first to fourth permanent premolars of the lower jaw are all absent when dog No. 220716, and an obvious intertooth area (diastea) appears between the anterior teeth and the molars, and the model dog with the clinical typical permanent tooth development disorder phenotype indicates that the model dog with the WNT10A gene function deletion mutation is successfully constructed.

The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.

Claims (14)

1. A method for establishing a model dog of a WNT10A gene deletion function variation disease comprises the step of obtaining a canine fertilized egg or canine somatic cell with reduced or deleted WNT10A gene expression by utilizing a gene editing technology.
2. 2. The method according to claim 1, wherein the gene editing technique is selected from BE3 single base editing technique, CRISPR, TALEN and ZFN, preferably CRISPR/Cas9.
3. 3. The method according to claim 1, characterized in that it comprises a targeted mutation of exon 2 of the WNT10A gene, preferably said mutation comprising an insertion, substitution or deletion of a nucleotide.
4. 4. The method according to claim 1, characterized in that it comprises the steps of:

   (1) Determining a target site according to the sequence of exon 2 of the canine WNT10A gene;

   (2) Synthesizing an sgRNA sequence according to the targeting site determined in the step (1), and then connecting the synthesized sequence with a skeleton carrier to construct an sgRNA targeting carrier;

   (3) In vitro transcription products of sgRNA and CRISPR/Cas9 are obtained by in vitro transcription, respectively;

   (4) Introducing the sgRNA and CRISPR/Cas9 in vitro transcription product obtained in the step (3) into a canine fertilized egg or canine somatic cell to obtain a canine fertilized egg or canine somatic cell with reduced or deleted WNT10A gene expression;

   preferably, in the step (1), 3 sgRNAs are determined based on the sequence of exon 2 of the canine WNT10A gene,

   more preferably, the sequence of the sgRNA and its complement comprises the following sequences:

   sgRNA1：GCGGAGGCCCAGAATGTCGTTGG(SEQ ID NO:2)

   complementary sequence of sgRNA 1: CCAACGACATTCTGGGCCTCCGC (SEQ ID NO: 3)

   sgRNA2：CAACGTTAGGCACACCGTGTTGG(SEQ ID NO:4)

   Complementary sequence of sgRNA 2: CCAACACGGTGTGCCTAACGTTG (SEQ ID NO: 5)

   sgRNA3：GGCACTCATGGATGGCGATCTGG(SEQ ID NO:6)

   Complementary sequence of sgRNA 3: CCAGATCGCCATCCATGAGTGCC (SEQ ID NO: 7).
5. 5. The method of claim 1, further comprising transplanting the canine fertilized egg with reduced or deleted WNT10A gene expression into the oviduct of a recipient female canine, thereby preparing a model canine for WNT10A gene deletion functional variation disease; or the method further comprises the steps of transplanting the cell nucleus of the canine somatic cell with reduced or deleted WNT10A gene expression into the canine enucleated oocyte, and then transplanting the nuclear-transplanted canine enucleated oocyte into the oviduct of a recipient female canine, thereby preparing a model canine for the WNT10A gene deletion function variation disease.
6. 6. The method of claim 1, wherein the WNT10A gene deletion function variant disease model canine expresses a protein having an amino acid sequence as set forth in at least one of SEQ ID NOs 11, 13, 14, 15, and 19; and/or

   The genome of the WNT10A gene deletion function variation disease model dog comprises a nucleotide sequence shown as at least one of SEQ ID NOs 10, 12, 14, 16 and 18.
7. 7. Canine somatic cells, tissues or organs of a model canine for a WNT10A gene-deleted functional variant disease obtained by the method of any one of claims 1-6;

   preferably, the canine somatic cell, tissue or organ expresses a protein having an amino acid sequence as set forth in at least one of SEQ ID NOs 11, 13, 14, 15 and 19; and/or

   The genome of the canine somatic cell, tissue or organ comprises a nucleotide sequence as set forth in at least one of SEQ ID NOs 10, 12, 14, 16 and 18.
8. 8. A canine somatic cell of a canine model of a WNT10A gene-deleted functional variant disease, the genome of the canine somatic cell comprising a nucleotide sequence as set forth in at least one of SEQ ID NOs 10, 12, 14, 16, and 18.
9. 9. The targeting vector comprises an sgRNA sequence and a skeleton vector, wherein the sgRNA sequence is designed for a targeting site sequence determined by a No. 2 exon of the canine WNT10A gene;

   preferably, the sgrnas and their complements include the following sequences:

   sgRNA1：GCGGAGGCCCAGAATGTCGTTGG(SEQ ID NO:2)

   complementary sequence of sgRNA 1: CCAACGACATTCTGGGCCTCCGC (SEQ ID NO: 3)

   sgRNA2：CAACGTTAGGCACACCGTGTTGG(SEQ ID NO:4)

   Complementary sequence of sgRNA 2: CCAACACGGTGTGCCTAACGTTG (SEQ ID NO: 5)

   sgRNA3：GGCACTCATGGATGGCGATCTGG(SEQ ID NO:6)

   Complementary sequence of sgRNA 3: CCAGATCGCCATCCATGAGTGCC (SEQ ID NO: 7).
10. 10. A cell comprising the targeting vector of claim 9.
11. 11. The cell of claim 10, wherein the cell is incapable of developing into an animal.
12. 12. A primer pair comprising the sequence:

    forward primer: GTAACCCTTCCCCCAGATGC (SEQ ID NO: 8),

    reverse primer: CTCCCTCCCATTTCCGACAC (SEQ ID NO: 9).
13. 13. Use of a primer pair according to claim 12 in the detection of a WNT10A gene deletion function variant disease model dog comprising a genomic sequence of nucleotide sequences set forth in at least one of SEQ ID NOs 10, 12, 14, 16 and 18.
14. 14. Use of the WNT10A gene deletion function variation disease model canine obtained by the method of any one of claims 1-6 in screening for a medicament for preventing or treating permanent tooth dysplasia diseases.