CN114752623B

CN114752623B - Construction method and application of animal model of retinal pigment degeneration disease

Info

Publication number: CN114752623B
Application number: CN202210009324.6A
Authority: CN
Inventors: 孙晓东; 万晓玲; 刘珊
Original assignee: Shanghai First Peoples Hospital
Current assignee: Shanghai First Peoples Hospital
Priority date: 2022-01-06
Filing date: 2022-01-06
Publication date: 2024-02-09
Anticipated expiration: 2042-01-06
Also published as: CN114752623A

Abstract

The invention discloses a construction method and application of a retinal pigment degeneration animal model, and relates to the technical field of genetic engineering. The construction method disclosed by the invention utilizes the CRISPR/Cas9 technology to mutate the Rho gene of the target animal, so that the encoded Rho protein has mutation. The invention obtains the disease animal model based on the L125R mutation of Rho gene for the first time, has the expression or symptoms of the retinal pigment degeneration, and provides an animal model foundation for the pathophysiological mechanism research of the retinal pigment degeneration, the screening of related gene therapy drug research and the like.

Description

Construction method and application of animal model of retinal pigment degeneration disease

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a construction method and application of a retinal pigment degeneration animal model.

Background

The retinol intracellular opsin binds to 11-cis retinal formed by oxidation of vitamin a to form Rho. Under illumination, 11-cis-retinal is converted to all-trans-retinal, which is the first step in visual formation. Rho mutations often lead to retinal pigment degeneration (retinitis pigmentosa, RP) inherited by autosomal dominant species. RP is a common hereditary retinal degenerative disease worldwide, and is mainly represented by progressive loss of retinal rod cells and cone cells, and causes serious vision disorders, ultimately leading to binocular blindness; it not only affects the life and quality of life of the patient himself, but also brings great mental and economic burden to family members. Due to the heterogeneity of RP inheritance, patients also vary in age of onset, rate of progression, and genetic pattern. At present, the pathophysiological mechanism of the disease is not completely elucidated, and RP therapies which are in progress or have completed clinical trials are mainly gene therapy. However, no RP animal model based on Rho c.374T > G mutation site is established.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a construction method and application of a retinitis pigmentosa animal model. The animal model of the retinal pigment degeneration based on the Rho gene L125R mutation is obtained for the first time by the construction method of the invention, and the animal model of the disease has the expression or symptoms of the retinal pigment degeneration, thus providing an animal model foundation for the pathophysiological mechanism research of the retinal pigment degeneration, the screening of related gene therapy drugs and the like.

The invention is realized in the following way:

in one aspect, the invention provides a method for constructing a Rho gene mutation-based retinal pigment degeneration animal model, which utilizes CRISPR/Cas9 technology to mutate the Rho gene of a target animal: 374T > G, thereby providing the encoded Rho protein with a mutation: L125R.

The mutation site is point mutation, and is very suitable for gene therapy. Rho gene is a pathogenic mutation gene of RP, and based on the disease animal model prepared by the Rho gene, the Rho gene is very important for researching the pathophysiological mechanism of RP and developing an effective treatment method and medicine of RP.

The animal model of the retinitis pigmentosa based on Rho gene mutation is obtained for the first time by the construction method of the invention, and the animal model of the disease has the expression or symptoms of the retinitis pigmentosa, thus providing a model foundation for researching pathophysiological mechanisms of the retinitis pigmentosa, screening related gene therapeutic drugs and the like.

Alternatively, in some embodiments, the target animal is a mouse or a rat. In other embodiments, those skilled in the art can readily select other mammals to construct animal models based on the present disclosure, which are also within the scope of the present invention.

Optionally, in some embodiments, the method of mutating the Rho gene of the target animal using CRISPR/Cas9 technology comprises:

microinjection of Cas9mRNA, gRNA, and homologous recombination vector into fertilized eggs of donor mice to obtain transfected fertilized eggs; wherein the gRNA is shown as SEQ ID NO. 1.

The CRISPR/Cas9 technology is adopted to edit the target gene, the gRNA is an important dependence of the gene editing effect, and the gene editing effect can not be realized by any gRNA. According to the invention, through creative labor, SEQ ID NO.1 is selected as a target sequence of gRNA, the target mutation of Rho genes can be effectively realized under the conditions that appropriate Cas9mRNA and appropriate homologous recombination vectors exist at target sites: 374T > G, thereby obtaining an animal model of Rho protein having the L125R mutation, and showing the corresponding symptoms of RP disease.

Alternatively, in some embodiments, the nucleic acid sequence of Cas9mRNA is set forth in SEQ ID No. 2.

Alternatively, in some embodiments, the homologous recombination vector comprises a homologous recombination fragment as set forth in SEQ ID NO. 3. The homologous recombination fragment comprises a 5 '-end homology arm, a Rho gene mutation sequence and a 3' -end homology arm which are sequentially connected in series. The animal model can be made to better mimic the function and effect of human Rho protein by introducing humanized Rho gene sequences.

Optionally, in some embodiments, the method of mutating a Rho gene of the target animal using CRISPR/Cas9 technology further comprises: transplanting the transfected fertilized egg into the uterus of a pseudopregnant female mouse to obtain an F0 generation mouse; and identifying F0 generation mice to obtain chimeric mice positive for homologous recombination.

Optionally, in some embodiments, the method of mutating a Rho gene of the target animal using CRISPR/Cas9 technology further comprises: and mating the chimeric mice with wild mice, identifying the obtained offspring mice, mating F1 generation mice with homologous recombination positive, and identifying the obtained F2 generation mice to obtain heterozygote mice or homozygote mice, namely the retinitis pigmentosa animal model.

In another aspect, the present invention provides a method for breeding an animal model of retinitis pigmentosa, comprising: the animal models of retinal degenerative diseases obtained by the construction method according to any one of the above claims are mated with each other.

After obtaining the retinitis pigmentosa animal model by the aforementioned construction method, a person skilled in the art can obtain a large number of animal models based on the ordinary breeding or propagation method, which is easy to achieve for a person skilled in the art, and therefore, the use of such propagation method is also within the scope of the present invention.

In a further aspect, the present invention provides the use of an animal model of retinitis pigmentosa obtained by the construction method according to any of the preceding claims in a retinitis pigmentosa study aimed at the diagnosis or treatment of a non-disease.

In another aspect, the present invention provides the use of an animal model of retinitis pigmentosa obtained by the construction method described in any one of the above for screening a gene therapy drug for retinitis pigmentosa.

The retinitis pigmentosa animal model obtained by the above-described construction method has typical RP symptoms, and can be applied to various fields of research, such as pathophysiological mechanism research, drug screening research, especially screening research of gene therapy drugs, etc., which are easy to be implemented by those skilled in the art based on the present disclosure, and therefore, the retinitis pigmentosa animal model of the present invention belongs to the scope of protection of the present invention no matter what field the retinitis pigmentosa animal model is applied to.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram showing the construction strategy of an animal model of retinitis pigmentosa in example 1.

FIG. 2 is a plasmid map of the homologous recombination vector.

FIG. 3 is an electrophoretogram of homologous recombination vector enzyme digestion identification; 1: the NheI enzyme digestion identification result shows that the theoretical band size is 7475bp, 3650bp and 1240bp; m:1kb DNA ladder.

FIG. 4 is a schematic diagram of the identification strategy of F0 and F1 mice; homologous recombination positive mouse PCR identification protocol: the 5' arm homologous recombination positive genome should amplify a 4.8kb fragment, and the negative genome has no product; the 3' arm homologous recombination positive genome should amplify a 3.4kb fragment and the negative genome should amplify a 7.0kb fragment.

FIG. 5 is a PCR identification electrophoretogram of homologous recombination-positive F0 mice; number 45: f0 mice were numbered; WT: a wild-type control; m is a 1kb DNA marker.

FIG. 6 is a diagram of PCR identification electrophoresis of 5 'homology arms and 3' homology arms of F1 mice; the number: f1 generation mice were numbered; WT: a wild-type control; m:1kb DNA ladder.

FIG. 7 is a diagram of PCR identification of a homologous recombination positive F2 generation partial mouse; the number: f2 mice numbered; m Marker, takara DL2000 DNA Marker 3427A.

FIG. 8 shows ONL detection results for WT, heterozygote and homozygous mice;

a: representative HE staining images (eye sections in the 500-1000 μm region of the optic nerve) of WT, heterozygote (L125R/+) and homozygous (L125R/L125R) mice. The IS/OS and ONL layers were significantly thinner at 8 months of heterozygote compared to WT, and the IS/OS and ONL layers were significantly thinner at 2 months of homozygote compared to WT, and thinner at 3 months of homozygote. Scale bar: 20 μm.

B: measurement of ONL layer thickness in HE staining images of WT, heterozygote and homozygous mice. The ONL layer thickness of 8 month old heterozygotes and homozygous (2 months and 3 months old) mice was significantly lower than WT.

C: heterozygote (3 months and 8 months old) and homozygote (3 months old) mice represent fundus images and corresponding OCT images. OCT is the image at the horizontal line on the corresponding fundus image, the ONL thickness of the heterozygote mice with 8 months of age (marked by white straight lines in OCT) is obviously lower than that of the heterozygote mice with 3 months of age, and the ONL thickness of the heterozygote mice with 3 months of age is obviously lower than that of the heterozygote mice with 8 months of age, which is consistent with HE results. RPE/CC: retinal pigment epithelial cells/choriocapillaris (Retinal Pigment Epithelium/choriocapillaries); IS/OS: inner/outer segments (inner/outer segments) of retinal photoreceptor cells; ONL: a photoreceptor outer nuclear layer (outer nuclear layer); INL: a core layer (inner nuclear layer). Scale bar: 120 μm.

FIG. 9 is a statistical analysis of ERG results for different time periods in WT, heterozygote and homozygous mice. The amplitude of the a wave and the b wave of the dark adaptation and the bright adaptation of the WT mice are not obviously different from each other between 6 weeks and 32 weeks, and the amplitude of the a wave and the b wave of the dark adaptation of heterozygote mice and homozygote mice is reduced along with the age. Homozygous mice varied significantly from 6 weeks, while heterozygotes were statistically different at 32 weeks. The clear adaptation a and b wave amplitudes of heterozygote and homozygote mice also decrease with age, but are not statistically significant. Dark adaptation a, b wave amplitudes represent rod cell function, while bright adaptation a, b wave amplitudes represent cone cell function. Both heterozygote and homozygote mice had obvious impairment of rod cell function, consistent with clinical manifestations of RP patients. 3-7 mice per group. * P <0.05; * P <0.005; * P <0.001.

FIG. 10 shows the western blot results of WT, heterozygote and homozygous mice 3 months old. The left side of the upper graph shows the expression of HA in three groups of retina, and the result of HA expression shows the content of mutant protein because the mutant sequence contains HA tag. The right is the expression of total rhodopsin, including muteins and normal mouse rhodopsin. The lower panel shows the expression of GAPDH (reference protein). The expression results of HA show that the content of mutant rhodopsin of homozygote is obviously higher than that of heterozygote, and WT does not contain mutant protein. For the total rhodopsin content, homozygotes are expressed significantly lower than homozygotes and WT of the same age.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The method for constructing the retinitis pigmentosa animal model provided in this embodiment adopts the CRISPR/Cas9 technology, and uses a homologous recombination mode to knock in the expression frame of hrno-HA-WPRE-pA at the initial codon site of the Rho gene (transcript: rho-201 (ensmest 00000032471.8)), see fig. 1. The hRho is a human Rho mutant gene sequence (c.374T > G; p.L125R), i.e., base T at position 374 is replaced by base G as compared to the reference sequence; the 125 th amino acid of the encoded protein is changed from leucine (L) to arginine (R). The method specifically comprises the following steps:

(1) A homologous recombination vector (donor vector) comprising a homologous recombination fragment having a 3.0kb 5 '-end homology arm, a hRho-HA-WPRE-pA and a 3.0kb 3' -end homology arm was constructed by an In-Fusion cloning method, the vector map is shown In FIG. 2, and the restriction map of the homologous recombination vector is shown In FIG. 3.

The nucleotide sequence of the homologous recombination fragment (SEQ ID NO. 3) is as follows:

wherein, 5' homology arm: bits 1-3000 (underlined);

kozak sequence: 3001 to 3006;

hRho-HA: 3007 th to 4110 th bits (lowercase); wherein, the box is the mutation site (c.374T > G);

WPRE: bits 4111-4698 (capital + underlined);

pA (Bgh polyA): bits 4714-4940 (capital + italics);

3' -homology arm: bits 4987-7986 (underlined).

(2) Cas9mRNA and gRNA were obtained by means of in vitro transcription, and Cas9mRNA, gRNA and donor vector were microinjected into fertilized eggs of donor mice (C57 BL/6J) to obtain transfected fertilized eggs. The gRNA sequence is 5'-tcgagagccgcagccatgaa-3' (SEQ ID NO. 1).

The nucleotide sequence of Cas9mRNA (SEQ ID No. 2) is as follows (underlined as coding sequence):

gagacaagcuugcuuguucuuuuugcagaagcucagaauaaacgcucaacuuuggcagaucgguaccgccaccauggacuauaaggaccacgacggagacuacaaggaucaugauauugauuacaaagacgaugacgauaagau ggccccaaagaagaagcggaaggucgguauccacggagucccagcagccgacaagaaguacagcaucggccuggac aucggcaccaacucugugggcugggccgugaucaccgacgaguacaaggugcccagcaagaaauucaaggugcugg gcaacaccgaccggcacagcaucaagaagaaccugaucggagcccugcuguucgacagcggcgaaacagccgaggc cacccggcugaagagaaccgccagaagaagauacaccagacggaagaaccggaucugcuaucugcaagagaucuuc agcaacgagauggccaagguggacgacagcuucuuccacagacuggaagaguccuuccugguggaagaggauaaga agcacgagcggcaccccaucuucggcaacaucguggacgagguggccuaccacgagaaguaccccaccaucuacca ccugagaaagaaacugguggacagcaccgacaaggccgaccugcggcugaucuaucuggcccuggcccacaugauc aaguuccggggccacuuccugaucgagggcgaccugaaccccgacaacagcgacguggacaagcuguucauccagc uggugcagaccuacaaccagcuguucgaggaaaaccccaucaacgccagcggcguggacgccaaggccauccuguc ugccagacugagcaagagcagacggcuggaaaaucugaucgcccagcugcccggcgagaagaagaauggccuguuc ggaaaccugauugcccugagccugggccugacccccaacuucaagagcaacuucgaccuggccgaggaugccaaac ugcagcugagcaaggacaccuacgacgacgaccuggacaaccugcuggcccagaucggcgaccaguacgccgaccu guuucuggccgccaagaaccuguccgacgccauccugcugagcgacauccugagagugaacaccgagaucaccaag gccccccugagcgccucuaugaucaagagauacgacgagcaccaccaggaccugacccugcugaaagcucucgugc ggcagcagcugccugagaaguacaaagagauuuucuucgaccagagcaagaacggcuacgccggcuacauugacgg cggagccagccaggaagaguucuacaaguucaucaagcccauccuggaaaagauggacggcaccgaggaacugcuc gugaagcugaacagagaggaccugcugcggaagcagcggaccuucgacaacggcagcaucccccaccagauccacc ugggagagcugcacgccauucugcggcggcaggaagauuuuuacccauuccugaaggacaaccgggaaaagaucga gaagauccugaccuuccgcauccccuacuacgugggcccucuggccaggggaaacagcagauucgccuggaugacc agaaagagcgaggaaaccaucacccccuggaacuucgaggaagugguggacaagggcgcuuccgcccagagcuuca ucgagcggaugaccaacuucgauaagaaccugcccaacgagaaggugcugcccaagcacagccugcuguacgagua cuucaccguguauaacgagcugaccaaagugaaauacgugaccgagggaaugagaaagcccgccuuccugagcggc gagcagaaaaaggccaucguggaccugcuguucaagaccaaccggaaagugaccgugaagcagcugaaagaggacu acuucaagaaaaucgagugcuucgacuccguggaaaucuccggcguggaagaucgguucaacgccucccugggcac auaccacgaucugcugaaaauuaucaaggacaaggacuuccuggacaaugaggaaaacgaggacauucuggaagau aucgugcugacccugacacuguuugaggacagagagaugaucgaggaacggcugaaaaccuaugcccaccuguucg acgacaaagugaugaagcagcugaagcggcggagauacaccggcuggggcaggcugagccggaagcugaucaacgg cauccgggacaagcaguccggcaagacaauccuggauuuccugaaguccgacggcuucgccaacagaaacuucaug cagcugauccacgacgacagccugaccuuuaaagaggacauccagaaagcccagguguccggccagggcgauagcc ugcacgagcacauugccaaucuggccggcagccccgccauuaagaagggcauccugcagacagugaagguggugga cgagcucgugaaagugaugggccggcacaagcccgagaacaucgugaucgaaauggccagagagaaccagaccacc cagaagggacagaagaacagccgcgagagaaugaagcggaucgaagagggcaucaaagagcugggcagccagaucc ugaaagaacaccccguggaaaacacccagcugcagaacgagaagcuguaccuguacuaccugcagaaugggcggga uauguacguggaccaggaacuggacaucaaccggcuguccgacuacgauguggaccauaucgugccucagagcuuu cugaaggacgacuccaucgacaacaaggugcugaccagaagcgacaagaaccggggcaagagcgacaacgugcccu ccgaagaggucgugaagaagaugaagaacuacuggcggcagcugcugaacgccaagcugauuacccagagaaaguu cgacaaucugaccaaggccgagagaggcggccugagcgaacuggauaaggccggcuucaucaagagacagcuggug gaaacccggcagaucacaaagcacguggcacagauccuggacucccggaugaacacuaaguacgacgagaaugaca agcugauccgggaagugaaagugaucacccugaaguccaagcugguguccgauuuccggaaggauuuccaguuuua caaagugcgcgagaucaacaacuaccaccacgcccacgacgccuaccugaacgccgucgugggaaccgcccugauc aaaaaguacccuaagcuggaaagcgaguucguguacggcgacuacaagguguacgacgugcggaagaugaucgcca agagcgagcaggaaaucggcaaggcuaccgccaaguacuucuucuacagcaacaucaugaacuuuuucaagaccga gauuacccuggccaacggcgagauccggaagcggccucugaucgagacaaacggcgaaaccggggagaucgugugg gauaagggccgggauuuugccaccgugcggaaagugcugagcaugccccaagugaauaucgugaaaaagaccgagg ugcagacaggcggcuucagcaaagagucuauccugcccaagaggaacagcgauaagcugaucgccagaaagaagga cugggacccuaagaaguacggcggcuucgacagccccaccguggccuauucugugcuggugguggccaaaguggaa aagggcaaguccaagaaacugaagagugugaaagagcugcuggggaucaccaucauggaaagaagcagcuucgaga agaaucccaucgacuuucuggaagccaagggcuacaaagaagugaaaaaggaccugaucaucaagcugccuaagua cucccuguucgagcuggaaaacggccggaagagaaugcuggccucugccggcgaacugcagaagggaaacgaacug gcccugcccuccaaauaugugaacuuccuguaccuggccagccacuaugagaagcugaagggcucccccgaggaua augagcagaaacagcuguuuguggaacagcacaagcacuaccuggacgagaucaucgagcagaucagcgaguucuc caagagagugauccuggccgacgcuaaucuggacaaagugcuguccgccuacaacaagcaccgggauaagcccauc agagagcaggccgagaauaucauccaccuguuuacccugaccaaucugggagccccugccgccuucaaguacuuug acaccaccaucgaccggaagagguacaccagcaccaaagaggugcuggacgccacccugauccaccagagcaucac cggccuguacgagacacggaucgaccugucucagcugggaggcgacaaaaggccggcggccacgaaaaaggccggc caggcaaaaaagaaaaagugaucuagugacugacuaggaucugguuaccacuaaaccagccucaagaacacccgaauggagucucuaagcuacauaauaccaacuuacacuuuacaaaauguugucccccaaaauguagccauucguaucugcuccuaauaaaaagaaaguuucuucacauucuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa。

(3) Transplanting the transfected fertilized eggs into the uterus of a pseudopregnant female mouse (C57 BL/6J), wherein the mice born for about 20 days are F0-generation mice; performing PCR amplification and sequencing identification on the F0 generation mice, wherein the mice with positive homologous recombination are chimeric mice; schematic of F0 and F1 generation mice identification strategy is shown in fig. 4. Homologous recombination positive mouse PCR identification protocol: the 5' arm homologous recombination positive genome should amplify a 4.8kb fragment, and the negative genome has no product; the 3' arm homologous recombination positive genome should amplify a 3.4kb fragment and the negative genome should amplify a 7.0kb fragment.

The primer sequences of the 5 'arm adopted by the PCR identification method are I and II, the primer sequences of the 3' arm adopted by the PCR identification method are III and IV, the genome DNA is extracted from the tail or the toe of the rat, and the nucleotide sequences of the primers are shown in the following table:

primer(s)	Sequence 5'-3'	Primer type
			I	TGCCGGCCAACCAGACTCATA	Upstream of
II	AACGGGCCACAACTCCTCATAAAG	Downstream of
			III	TTTGCTAGCTCCGTTTCCACATTG	Upstream of
IV	GTCCCATTCTCCCCCTCCCCTTCT	Downstream of

The reaction system of the F0 generation PCR identification method is as follows:

the reaction conditions of the F0 generation PCR identification method are as follows:

Step#	Temp(℃)	Time	Note
				1	94	3min	-
2	98	15sec	-
				3	61	15sec	-
4	68	3min	repeat steps 2-4for 35cycles
				5	68	5min	-
6	12	-	hold

the results are shown in FIG. 5: the 5' arm homologous recombination positive genome should amplify a 4.8kb fragment, and the negative genome should be free of products; the 3' arm homologous recombination positive genome should amplify a 3.4kb fragment and the negative genome should amplify a 7.0kb fragment.

(4) Mating the chimeric mice with wild C57BL/6J mice to obtain offspring mice, and identifying the offspring mice, wherein the mice with positive homologous recombination are F1-generation mice;

the PCR identification method is adopted for F1 generation identification, the specific method is the same as F0 generation mouse identification method, and the result is shown in figure 6.

(5) The F1 generation mice are inbred with each other to obtain F2 generation mice, and heterozygote and homozygote mice in the mice are identified.

The PCR identification method is adopted for the F2 generation identification, and the primer pair (rho F1, rho R1) and the primer pair (rho F2, rho R2) are used for the F2 generation mice to amplify respectively, wherein the specific sequences are shown in the following table:

primer(s)	Sequence 5'-3'	Primer type
			rho F1	GGCAGCAGTGGGATTAGCGTTAGTATGA	Upstream of
rho R1	GCTTACACACCACCACGTACC	Downstream of
			rho F2	CCTTGGTCTCTGTCTACGAAGAGC	Upstream of
rho R2	GCCCCAGTCTCTCTGCTCATAC	Downstream of

Reaction system of PCR identification method:

the reaction conditions of the PCR identification method are as follows:

the results of the identification of the partial F2 mice are shown in fig. 7 and 8:

wild-type mice only (rho F2, rho R2) amplified a 435bp fragment, (rho F1, rho R1) were banded;

heterozygotes: (rho F1, rho R1) amplified a 633bp fragment and (rho F2, rho R2) amplified a 435bp fragment;

homozygote: (rho F1, rho R1) a 633bp fragment was amplified and (rho F2, rho R2) was nonreactive.

As can be seen in fig. 7, nos. 922 and 949 are homozygotes; 728F 1 generation, 971, 781, and 728 heterozygotes as positive controls.

Example 2

Heterozygote and homozygote mice of example 1 were phenotyped as follows:

(1) Fundus photo and Optical Coherence Tomography (OCT): experimental mice were anesthetized with 1% sodium pentobarbital and 1% topiramate mydriasis (Santen pharmaceutical, osaka, japan). 0.4% oxybuprocaine hydrochloride eye drops (Santen Pharmaceuticals, osaka, japan) were locally anesthetized. Fundus photographs and Optical Coherence Tomography (OCT) were performed using a micro IV system (Phoenix Research Laboratories, plaasanton, CA, USA).

(2) ERG: full field Electroretinograms (ERG) were recorded and retinal function was assessed using a report System (Roland condult, brandenburg, germany) and Super Color Ganzfeld (Q450 SC) stimulator. After dark adaptation overnight, the abdominal cavity was anesthetized with 1% sodium pentobarbital, 1% topicalamine, 0.4% oxybuprocaine hydrochloride eye drops for mydriasis and local anesthesia. Body temperature was maintained at 37 ℃ during the experiment and heated with an electric pad. The reference electrode is placed in the center of the scalp and the ground electrode is placed at the proximal end of the tail skin. Contact lens electrodes are applied directly to the corneal surface. The mice were placed in the center to ensure that both eyes were illuminated equally at the same time. All procedures were performed under dim red light. The light stimulus was fixed at a constant luminance (3 cd. Times.s/m ² ). The negative wave after the optical stimulus is defined as the a wave, and the positive peak is defined as the b wave.

(3) HE staining: each group was fixed with 3 eyes, 4% paraformaldehyde for 24h, paraffin embedded, and each slice was 5 μm thick. Staining with Hematoxylin and Eosin (HE). The ONL damage was evaluated by taking the average of the thickness of 3 ONL measurements every 200 μm from the optic disc.

(4) Western Blot: homogeneity was achieved with lysis buffer containing 50mmol/L Tris 7.4, 150mmol/L NaCl, 1% Triton X-100 (Sigma-Aldrich), 1% sodium deoxycholate, 0.1% SDS, protease inhibitor (Roche) and phosphatase inhibitor (Sigma-Aldrich). Samples were subjected to 8% -15% Bis-Tris gel electrophoresis and then electrotransferred to PVDF membrane. Incubation with 5% BSA with tris-buffered saline and 0.02% Tween-20 (pH 7.4; sigma-Aldrich) was performed for 1.5 hours at room temperature. PVDF membrane was incubated overnight at 4℃with HA (AH 158-1, beyotidme, 1:1000), rhodopsin (14825, CST, 1:1000) and GAPDH (10494-1-AP, proteintech, 1:2000) antibodies. After washing, the membranes were incubated with the corresponding horseradish peroxidase-labeled secondary antibodies for 1 hour at room temperature. Protein bands were observed using enhanced chemiluminescence techniques (Amersham Pharmacia Biotech, amersham, UK).

Each experiment was repeated at least three times. Data are expressed as mean ± SD. The differences between groups were tested using independent samples. All statistical analyses were performed using Prism 8.0 software (GraphPad software, la Jolla, calif., USA). P <0.05 was considered significant.

The results are shown in FIGS. 8-10; the OCT results of fig. 8C show that heterozygotes and homozygotes mice become older, photoreceptor cells IS/OS and ONL become thinner, homozygotes 3 months old ONL have been very thin, consistent with the HE staining results of fig. 8-a/B.

The results of fig. 9 show: homozygotes showed a marked impairment of rod cell function from 6 weeks; heterozygotes 32 showed impaired rod function and no apparent abnormality in cone function.

The results of fig. 10 show: the mutant sequence contains an HA tag, so the results of HA show that the homozygote mutant rhodopsin content is significantly higher than the heterozygote, and that WT does not contain a mutein. The expression of heterozygotes and WT total rhodopsin (including normal murine rhodopsin and muteins) is significantly higher than that of homozygote rhodopsin of the same age.

Taken together, it can be seen that the mouse model prepared in the examples of the present invention successfully expressed the human rho mutein carrying the HA tag. Homozygous mice showed a clear RP phenotype two months of birth and heterozygotes also showed photoreceptor cell damage at 8 months of birth. Therefore, the mouse model can be used for the research of pathophysiological mechanism of RP caused by Rho mutation, drug screening research, especially screening research of gene therapy drugs, and the like, and provides a reliable animal model foundation for the research.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Shanghai first people Hospital

<120> construction method and application of retinitis pigmentosa animal model

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

tcgagagccg cagccatgaa 20

<210> 2

<211> 4541

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

gagacaagcu ugcuuguucu uuuugcagaa gcucagaaua aacgcucaac uuuggcagau 60

cgguaccgcc accauggacu auaaggacca cgacggagac uacaaggauc augauauuga 120

uuacaaagac gaugacgaua agauggcccc aaagaagaag cggaaggucg guauccacgg 180

agucccagca gccgacaaga aguacagcau cggccuggac aucggcacca acucuguggg 240

cugggccgug aucaccgacg aguacaaggu gcccagcaag aaauucaagg ugcugggcaa 300

caccgaccgg cacagcauca agaagaaccu gaucggagcc cugcuguucg acagcggcga 360

aacagccgag gccacccggc ugaagagaac cgccagaaga agauacacca gacggaagaa 420

ccggaucugc uaucugcaag agaucuucag caacgagaug gccaaggugg acgacagcuu 480

cuuccacaga cuggaagagu ccuuccuggu ggaagaggau aagaagcacg agcggcaccc 540

caucuucggc aacaucgugg acgagguggc cuaccacgag aaguacccca ccaucuacca 600

ccugagaaag aaacuggugg acagcaccga caaggccgac cugcggcuga ucuaucuggc 660

ccuggcccac augaucaagu uccggggcca cuuccugauc gagggcgacc ugaaccccga 720

caacagcgac guggacaagc uguucaucca gcuggugcag accuacaacc agcuguucga 780

ggaaaacccc aucaacgcca gcggcgugga cgccaaggcc auccugucug ccagacugag 840

caagagcaga cggcuggaaa aucugaucgc ccagcugccc ggcgagaaga agaauggccu 900

guucggaaac cugauugccc ugagccuggg ccugaccccc aacuucaaga gcaacuucga 960

ccuggccgag gaugccaaac ugcagcugag caaggacacc uacgacgacg accuggacaa 1020

ccugcuggcc cagaucggcg accaguacgc cgaccuguuu cuggccgcca agaaccuguc 1080

cgacgccauc cugcugagcg acauccugag agugaacacc gagaucacca aggccccccu 1140

gagcgccucu augaucaaga gauacgacga gcaccaccag gaccugaccc ugcugaaagc 1200

ucucgugcgg cagcagcugc cugagaagua caaagagauu uucuucgacc agagcaagaa 1260

cggcuacgcc ggcuacauug acggcggagc cagccaggaa gaguucuaca aguucaucaa 1320

gcccauccug gaaaagaugg acggcaccga ggaacugcuc gugaagcuga acagagagga 1380

ccugcugcgg aagcagcgga ccuucgacaa cggcagcauc ccccaccaga uccaccuggg 1440

agagcugcac gccauucugc ggcggcagga agauuuuuac ccauuccuga aggacaaccg 1500

ggaaaagauc gagaagaucc ugaccuuccg cauccccuac uacgugggcc cucuggccag 1560

gggaaacagc agauucgccu ggaugaccag aaagagcgag gaaaccauca cccccuggaa 1620

cuucgaggaa gugguggaca agggcgcuuc cgcccagagc uucaucgagc ggaugaccaa 1680

cuucgauaag aaccugccca acgagaaggu gcugcccaag cacagccugc uguacgagua 1740

cuucaccgug uauaacgagc ugaccaaagu gaaauacgug accgagggaa ugagaaagcc 1800

cgccuuccug agcggcgagc agaaaaaggc caucguggac cugcuguuca agaccaaccg 1860

gaaagugacc gugaagcagc ugaaagagga cuacuucaag aaaaucgagu gcuucgacuc 1920

cguggaaauc uccggcgugg aagaucgguu caacgccucc cugggcacau accacgaucu 1980

gcugaaaauu aucaaggaca aggacuuccu ggacaaugag gaaaacgagg acauucugga 2040

agauaucgug cugacccuga cacuguuuga ggacagagag augaucgagg aacggcugaa 2100

aaccuaugcc caccuguucg acgacaaagu gaugaagcag cugaagcggc ggagauacac 2160

cggcuggggc aggcugagcc ggaagcugau caacggcauc cgggacaagc aguccggcaa 2220

gacaauccug gauuuccuga aguccgacgg cuucgccaac agaaacuuca ugcagcugau 2280

ccacgacgac agccugaccu uuaaagagga cauccagaaa gcccaggugu ccggccaggg 2340

cgauagccug cacgagcaca uugccaaucu ggccggcagc cccgccauua agaagggcau 2400

ccugcagaca gugaaggugg uggacgagcu cgugaaagug augggccggc acaagcccga 2460

gaacaucgug aucgaaaugg ccagagagaa ccagaccacc cagaagggac agaagaacag 2520

ccgcgagaga augaagcgga ucgaagaggg caucaaagag cugggcagcc agauccugaa 2580

agaacacccc guggaaaaca cccagcugca gaacgagaag cuguaccugu acuaccugca 2640

gaaugggcgg gauauguacg uggaccagga acuggacauc aaccggcugu ccgacuacga 2700

uguggaccau aucgugccuc agagcuuucu gaaggacgac uccaucgaca acaaggugcu 2760

gaccagaagc gacaagaacc ggggcaagag cgacaacgug cccuccgaag aggucgugaa 2820

gaagaugaag aacuacuggc ggcagcugcu gaacgccaag cugauuaccc agagaaaguu 2880

cgacaaucug accaaggccg agagaggcgg ccugagcgaa cuggauaagg ccggcuucau 2940

caagagacag cugguggaaa cccggcagau cacaaagcac guggcacaga uccuggacuc 3000

ccggaugaac acuaaguacg acgagaauga caagcugauc cgggaaguga aagugaucac 3060

ccugaagucc aagcuggugu ccgauuuccg gaaggauuuc caguuuuaca aagugcgcga 3120

gaucaacaac uaccaccacg cccacgacgc cuaccugaac gccgucgugg gaaccgcccu 3180

gaucaaaaag uacccuaagc uggaaagcga guucguguac ggcgacuaca agguguacga 3240

cgugcggaag augaucgcca agagcgagca ggaaaucggc aaggcuaccg ccaaguacuu 3300

cuucuacagc aacaucauga acuuuuucaa gaccgagauu acccuggcca acggcgagau 3360

ccggaagcgg ccucugaucg agacaaacgg cgaaaccggg gagaucgugu gggauaaggg 3420

ccgggauuuu gccaccgugc ggaaagugcu gagcaugccc caagugaaua ucgugaaaaa 3480

gaccgaggug cagacaggcg gcuucagcaa agagucuauc cugcccaaga ggaacagcga 3540

uaagcugauc gccagaaaga aggacuggga cccuaagaag uacggcggcu ucgacagccc 3600

caccguggcc uauucugugc uggugguggc caaaguggaa aagggcaagu ccaagaaacu 3660

gaagagugug aaagagcugc uggggaucac caucauggaa agaagcagcu ucgagaagaa 3720

ucccaucgac uuucuggaag ccaagggcua caaagaagug aaaaaggacc ugaucaucaa 3780

gcugccuaag uacucccugu ucgagcugga aaacggccgg aagagaaugc uggccucugc 3840

cggcgaacug cagaagggaa acgaacuggc ccugcccucc aaauauguga acuuccugua 3900

ccuggccagc cacuaugaga agcugaaggg cucccccgag gauaaugagc agaaacagcu 3960

guuuguggaa cagcacaagc acuaccugga cgagaucauc gagcagauca gcgaguucuc 4020

caagagagug auccuggccg acgcuaaucu ggacaaagug cuguccgccu acaacaagca 4080

ccgggauaag cccaucagag agcaggccga gaauaucauc caccuguuua cccugaccaa 4140

ucugggagcc ccugccgccu ucaaguacuu ugacaccacc aucgaccgga agagguacac 4200

cagcaccaaa gaggugcugg acgccacccu gauccaccag agcaucaccg gccuguacga 4260

gacacggauc gaccugucuc agcugggagg cgacaaaagg ccggcggcca cgaaaaaggc 4320

cggccaggca aaaaagaaaa agugaucuag ugacugacua ggaucugguu accacuaaac 4380

cagccucaag aacacccgaa uggagucucu aagcuacaua auaccaacuu acacuuuaca 4440

aaauguuguc ccccaaaaug uagccauucg uaucugcucc uaauaaaaag aaaguuucuu 4500

cacauucuaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 4541

<210> 3

<211> 7986

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

tccattcaat gccctgtgac tgcccctgct tctgaagggc caacatggct acagctagct 60

ccagagacag cttttcaggg ccccagcatc caagcatctc acagttctcc actgaccaca 120

ctcctgtgca gcactgggct tttcaatgcc cctgacttga agagaactca aactgcaggt 180

caactagact ctgcaaactt cacctgtgct gggggttcct agcctgtggg gacagtgtat 240

cttgaatacc tgctgctatg gaccaagagc tgaacacaca gacaaacagg ctcagctggc 300

cggcattctg gaaccacaaa tgagtgtgga tgagcaggag ggcaacaaaa tggtctgggt 360

gttgtcaaca cagtcagtaa acaatgcacg cagtggggct gggccctgat gtggagctag 420

gtggggttgg ctctccttgg aaacctgaag ggagaaggag agggagcgag atgatgaggt 480

ttatcagcct gcagaggcag ggggtcagga aggagtgcca ctgtactgac ccaggacctc 540

tgtgggacat caagccatgc caaggagcca tggagcctcg attgcactgg cagggacagg 600

ttgtgatgcc ccagagtccc cagacccagc aaacagaggc ccagagtggg aagtggagct 660

ttccagggta tcggggtgac tcagagacac agggtagaat ctgccttggg tgctcactgc 720

cctatctgag tccacatggc tcagtcccca ggccctgttc tctagtgact gttgctttga 780

tgaggtagag acaggcagcc ctcttctaag aactatgttt tgatggggga ctcagagttg 840

gggtggggtg gcaatgaaat tctgtagact gtgtggttat aaccctggct gttactagct 900

agttctgtga ccttggtgac ccacttcaga ctctaggcct cagcctctgt aagtgcagat 960

acacagcgcc aatcagccga tgacttctaa caatactctt aactcacaca gagcttgtct 1020

cactgagcca acaccctgta ccctcagctc agtgacggct ttcaacctgt ggggctgcct 1080

ctgttaccca agtgagagag ggccagtgct cccagaggtg accttgtttg cccattctct 1140

ccctgggtca gccagtgttt atctgttgta tacccagtcc accctgcagg ctcacatcag 1200

agcctaggag atggctagtg tccccgcgga gaccacgatg aagcttccca gctgtctcaa 1260

gcacaagctg gctgcagagg ctgctgaggc actgctagct ggggatgggg gcagggtaga 1320

tctggggctg accaccaggg tcagaatcag aacctccacc ttgacctcat taacgctggt 1380

cttaatcacc aagccaagct ccttaaactg ctagtggcca actcccaggc cctgacacac 1440

atacctgccc tgtgttccca aacaagacac ctgcatggaa ggaagggggt tgcttttcta 1500

agcaaacatc taggaatccc gggtgcagtg tgaggagact aggcgaggga gtactttaag 1560

ggcctcaagg ctcagagagg aatacttctt ccctggttag cctcgtgcct aggctccagg 1620

gtctttgtcc tgcctggata cctatgtggc aaggggcata gcatttcccc caccatcagc 1680

tcttagctca accttatctt ctcggaaaga ctgcgcagtg taacaacaca gcagagactt 1740

ttcttttgtc ccctgtctac ccctgtaact gctactcaga agcatctttc tcacagggta 1800

ctggcttctt gcatccagag ttttttgtct ccctcgggcc cccagaatca aattcttcct 1860

ctgggactca gtggatgttt cacacacgta tcggcctgac agtcatcctg gagcatccta 1920

cacaggggcc atcacagctg catgtcagaa atgctggcct cacatcctca gacaccaggc 1980

ctagtgctgg tcttcctcag actggcgtcc ccagcaggcc agtaggatca tcttttagcc 2040

tacagagttc tgaagcctca gagccccagg tccctggtca tcttctctgc ccctgagatt 2100

tttccaagtt gtatgccttc taggtaaggc aaaacttctt acgcccctcc tcgtggcctc 2160

caggccccac atgctcacct gaataacctg gcagcctgct ccctcatgca gggaccacgt 2220

cctgctgcac ccagcaggcc atcccgtctc catagcccat ggtcatccct ccctggacag 2280

gaatgtgtct cctccccggg ctgagtcttg ctcaagctag aagcactccg aacagggtta 2340

tgggcgcctc ctccatctcc caagtggctg gcttatgaat gtttaatgta catgtgagtg 2400

aacaaattcc aattgaacgc aacaaatagt tatcgagccg ctgagccggg gggcgggggg 2460

tgtgagactg gaggcgatgg acggagctga cggcacacac agctcagatc tgtcaagtga 2520

gccattgtca gggcttgggg actggataag tcagggggtc tcctgggaag agatgggata 2580

ggtgagttca ggaggagaca ttgtcaactg gagccatgtg gagaagtgaa tttagggccc 2640

aaaggttcca gtcgcagcct gaggccacca gactgacatg gggaggaatt cccagaggac 2700

tctggggcag acaagatgag acaccctttc ctttctttac ctaagggcct ccacccgatg 2760

tcaccttggc ccctctgcaa gccaattagg ccccggtggc agcagtggga ttagcgttag 2820

tatgatatct cgcggatgct gaatcagcct ctggcttagg gagagaaggt cactttataa 2880

gggtctgggg ggggtcagtg cctggagttg cgctgtggga gccgtcagtg gctgagctcg 2940

ccaagcagcc ttggtctctg tctacgaaga gcccgtgggg cagcctcgag agccgcagcc 3000

gccaccatga atggcacaga aggccctaac ttctacgtgc ccttctccaa tgcgacgggt 3060

gtggtacgca gccccttcga gtacccacag tactacctgg ctgagccatg gcagttctcc 3120

atgctggccg cctacatgtt tctgctgatc gtgctgggct tccccatcaa cttcctcacg 3180

ctctacgtca ccgtccagca caagaagctg cgcacgcctc tcaactacat cctgctcaac 3240

ctagccgtgg ctgacctctt catggtccta ggtggcttca ccagcaccct ctacacctct 3300

ctgcatggat acttcgtctt cgggcccaca ggatgcaatt tggagggctt ctttgccacc 3360

ctgggcggtg aaattgcccg gtggtccttg gtggtcctgg ccatcgagcg gtacgtggtg 3420

gtgtgtaagc ccatgagcaa cttccgcttc ggggagaacc atgccatcat gggcgttgcc 3480

ttcacctggg tcatggcgct ggcctgcgcc gcacccccac tcgccggctg gtccaggtac 3540

atccccgagg gcctgcagtg ctcgtgtgga atcgactact acacgctcaa gccggaggtc 3600

aacaacgagt cttttgtcat ctacatgttc gtggtccact tcaccatccc catgattatc 3660

atctttttct gctatgggca gctcgtcttc accgtcaagg aggccgctgc ccagcagcag 3720

gagtcagcca ccacacagaa ggcagagaag gaggtcaccc gcatggtcat catcatggtc 3780

atcgctttcc tgatctgctg ggtgccctac gccagcgtgg cattctacat cttcacccac 3840

cagggctcca acttcggtcc catcttcatg accatcccag cgttctttgc caagagcgcc 3900

gccatctaca accctgtcat ctatatcatg atgaacaagc agttccggaa ctgcatgctc 3960

accaccatct gctgcggcaa gaacccactg ggtgacgatg aggcctctgc taccgtgtcc 4020

aagacggaga cgagccaggt ggccccggcc tacccatacg atgttccaga ttacgctggc 4080

tacccatacg atgttccaga ttacgcttaa tcaacctctg gattacaaaa tttgtgaaag 4140

attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat 4200

gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc 4260

ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg 4320

cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct 4380

ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct 4440

tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg 4500

gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac 4560

gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct 4620

gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct 4680

ttgggccgcc tccccgcatc gataccgtcg atcctgtgcc ttctagttgc cagccatctg 4740

ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 4800

cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 4860

gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg 4920

atgcggtggg ctctatggga tttgctagct ccgtttccac attgccaact tcggccccat 4980

cttcatgtat gagcagagag actggggcgg gggggtgtag catgggagcc aaggggccac 5040

gaaagggcct gggagggtct gcagcttact tgagtctctt taattggtct catctaaagg 5100

cccagcttat tcattggcaa acactgtgac cctgagctag gctgctgttg agagcaggca 5160

cggaacattc atctatctca tcttgagcaa tgcaagaaac atgggttcag agaggccaag 5220

gactcaccga ggagtcacag agtgtggggg tgtcctctga ggcagctgag ctggggcaca 5280

cacagactga gcaccaggag tgagctctag cttttttttt tctatgtgtc ttttctaaaa 5340

gacacatagg tttaggactg tccctggtcc aggtaagaac tggttcagta aacttgtaca 5400

tctcactgcc tggccagccc tgtcagcttc caccagagtg cgtgcactac acacccggca 5460

tctcaaagga ttcattccta tctttcctat ctttggagtg aggcacagtc tcacgtagtc 5520

cagtccagac tggccttaaa ttctgcagct gaggatgtac ttaaacttgt catcctcctg 5580

ccccagcctc tcaagtgctg tgatcacagg cacggaccac tatgctacgc caggtgtttc 5640

caaacatttt ctctccctta actggaaggt caatgaggct ctttcgagaa gcaacagagc 5700

ctgtttagct gagaaaactg aggcagggag caggcaaaaa tcacatctag agatatggga 5760

gagccaggac cagagccagg gtctccgggc tgggacctag agatgtttcc agtggataca 5820

ggaggaaaca gaagtgggtg tagcaaagcc caaagccagg gtgatgggtg ggtcgagctt 5880

gcttatctcc ccgtgtccag ggtactgcct tggtagcact gttgggcatc tctgttacct 5940

tctgtggtca cacacacaca cacacacaca cacacacaca cacacacaca tacacacaca 6000

cacacccagg ttctggatag aagctggggt accatgccgg tgagcttgtc tctgtggggt 6060

cagacccagg ccacatctac catccaggat tcttgttggt agcactcctg ttattcagaa 6120

gtttgttacg tggtccttcc ccacactggg cttctgaggc tgacatatgg actaatgtct 6180

ggagccccct ctttacccat cttcttcccc tgctaaaaca ctagccaggg tgtggcccta 6240

agccccagct ccaggcactc cgaggcagtc tctcatgagc ctaaagctct aaccaaacag 6300

aagagcttct gttttggcac acgggttctt caccccatcc ctttctcctc gccagcccaa 6360

actcactgca gtcgctaagg cttggatcaa gcctcaaacc agaagcttgc atcctagcct 6420

gctctctctg aggtgaggtt agagctggag gactgacggc tactaactgc cttacaggtg 6480

aaatcgccct gtggtccctg gtggtcctgg ccattgagcg ctacgtggtg gtctgcaagc 6540

cgatgagcaa cttccgcttc ggggagaatc acgctatcat gggtgtggtc ttcacctgga 6600

tcatggcgtt ggcctgtgct gctcccccac tcgttggctg gtccaggtaa tggcactgag 6660

tatcgggtct ggcaaggtct ttgtgggatt ccctttgagg acacagagcc ctcggattgg 6720

ttccaggcat aatgtaacat ggtattgccc cccgaaaacc atcctggtga ctttcccagg 6780

ctaaggtcta aggtagggga gaagagaggg actgaatggt ccaatcagtc ttattccatg 6840

tctgagaccc ataacaagga gaaccctgga cattccaacc cttcaccttg gccgagtccc 6900

taatcctcgg ctaagccaag gccaaaccac aatcctcttt ggttgagttc tggccgtggg 6960

cctctctctc tcttcctctc tctctctcac tcaccttgga ccttagcccc tggagaggct 7020

gaaccttccc aaaatgcatg gtgacattgt agccccagga actgggtccc atccagcctc 7080

caggccacca tatctaaatg agacaagaga aggttgggac agtggtttgg acacctagac 7140

aggctatgag gtacacagag cctcagagac tctccattct ctgtcctcat gtcctcccca 7200

ccccgggagc ccacccagtg gccctcactg agtcagagcc cctcactcct cactggcctc 7260

tttcctcatc ctcacccacc tggcttggca ggcctctaca gacacactca gtggacactt 7320

gggtttctga gtgtggccca gtgtcaccgt cctcagatat catcacaaca tccttgtttc 7380

tagaagctgc acacagccct gatgccagca gcgagcccac ctttactgta gagagcattg 7440

ccgttactag gagatcccat gcacaaagtg cagcattccc agggaaggcc tcagagaatg 7500

ctcctctctc cagcattctc tgcctactcc cttaaccacc gaaggcaggg cagcaggcta 7560

gtggagcaga gctgcgtggt caagtggcag ggagcttaag aatcgtccaa gggcggagac 7620

cagtaagtct cattaggtga tggggccagc aggtaaaagc cattcatgct tatgtccagc 7680

tgggcgtgtg ttctcttcct gttttatcat cccttgcgct gaccatcagg tacatccctg 7740

agggcatgca atgttcatgc gggattgact actacacact caagcctgag gtcaacaacg 7800

aatcctttgt catctacatg ttcgtggtcc acttcaccat tcctatgatc gtcatcttct 7860

tctgctatgg gcagctggtc ttcacagtca aggaggtatg agcagggggg ccgccccagc 7920

ctcgtgccgg gtgggtgggc agagccaggt gggcagagct gggtgccagg gttcgtacag 7980

acgcca 7986

Claims

1. A construction method of a retinoic pigment degeneration animal model based on Rho gene mutation is characterized in that the Rho gene of a target animal is mutated by using a CRISPR/Cas9 technology: 374T > G, thereby mutating the encoded Rho protein: L125R; the target animal is a mouse;

the method for mutating Rho gene of target animal by CRISPR/Cas9 technology includes:

microinjection of Cas9mRNA, gRNA, and homologous recombination vector into fertilized eggs of donor mice to obtain transfected fertilized eggs; wherein, the gRNA is shown as SEQ ID NO. 1;

the nucleic acid sequence of the Cas9mRNA is shown as SEQ ID NO. 2;

the homologous recombination vector contains homologous recombination fragments shown in SEQ ID NO. 3.

2. The method of construction of claim 1, wherein the method of mutating the Rho gene of the target animal using CRISPR/Cas9 technology further comprises: transplanting the transfected fertilized eggs into the uterus of pseudopregnant female mice to obtain F0 generation mice; and identifying F0 generation mice to obtain chimeric mice positive for homologous recombination.

3. The method of construction of claim 2, wherein the method of mutating the Rho gene of the target animal using CRISPR/Cas9 technology further comprises: and mating the chimeric mice with wild mice, identifying the obtained offspring mice, mating F1 generation mice with homologous recombination positive, and identifying the obtained F2 generation mice to obtain heterozygote mice or homozygote mice, namely the retinitis pigmentosa animal model.

4. A method of breeding an animal model of retinitis pigmentosa comprising: mating the animal models of retinitis pigmentosa disease obtained by the construction method of any of claims 1-3 with each other.

5. Use of an animal model of retinitis pigmentosa obtained by the construction method according to any of claims 1-3 in a retinitis pigmentosa study aimed at the diagnosis or treatment of a non-disease.

6. Use of an animal model of retinitis pigmentosa obtained by the construction method of any one of claims 1-3 for screening a gene therapy drug for retinitis pigmentosa.