CN113621649B - Construction method and application of retinal pigment degeneration disease model - Google Patents
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Abstract
The invention discloses a construction method and application of a retinal pigment degeneration disease model, and relates to the field of medical engineering. The construction method comprises the following steps: a model of a retinitis pigmentosa disease, which exhibits characteristics associated with the retinitis pigmentosa disease, can be obtained by preventing or inhibiting expression of the Ythdf1 gene sequence in the genome of the target animal. Such as rod cell death, is mainly manifested by photoreceptor damage, degeneration, progressive thinning of the outer retinal nuclear layer until disappearance, and corresponding pathological changes in the outer retinal network and other relevant cell layers. Therefore, the novel model of the retinal pigment degeneration disease can be used in the fields of research of the retinal pigment degeneration disease and the like, and provides a richer model selection for research of the disease such as pathogenesis, mechanism and screening of related drugs. In addition, the development of the disease model provides a new target for the treatment or prevention of RP diseases.
Description
Technical Field
The invention relates to the technical field of medical engineering, in particular to a construction method and application of a retinal pigment degeneration disease model.
Background
Retinitis pigmentosa (Retinitis pigmentosa, RP). RP as a hereditary blinding fundus disease can be divided into two categories according to clinical phenotype: typical RP patients and atypical RP patients. Wherein, typical RP patients show common damage of the rod cells, accounting for 80% -90% of the RP patients; atypical RP patients mainly have damaged cone cells, accounting for 10% -20% of RP patients. Typical RP patients develop night blindness and progressive visual field impairment, which develop tubular vision, at the earliest, due to defects in rod cell function, until blindness. In pathology, typical RP affects mainly rod cells, causing rod cell death and secondary cone cell death, manifested by photoreceptor cell damage, degeneration, progressive thinning of the outer nuclear layer of the retina until disappearance, with corresponding pathological changes in other related cell layers of the retina.
RP has incidence rate of 1/3500 in Chinese population, and RP patients can reach hundreds of thousands of people because of the population of China, thus bringing heavy burden to families and society. At present, diagnosis and treatment of RP still face many difficulties, mainly due to their high degree of heterogeneity in clinical phenotypes and genetics, and the lack of systematic studies on their pathogenesis, leading to an unclear specific molecular mechanism of RP.
At present, a corresponding RP disease model is lacking.
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 retinal pigment degeneration disease model to solve the technical problems.
YTDDF 1 (YTH N6-methyladenosine RNA binding protein 1, MGI: 1917431), this gene is located on mouse chromosome 2 at 180546170-180562729bp, full length 16.56kb, and its cDNA at 3199bp, contains 5 exons.
YTDDF 1 is taken as a reading protein of m6A methylation in RNA, can identify the methylation site of the RNA and further regulate and control, and is widely distributed in various tissue cells. m6A methylation is a dynamic reversible process, the formation process mainly comprises a methyltransferase complex (METTL 3, METTL14, WTAP and the like), the demethylation process is completed by demethylases (FTO and ALKBH 5), and reading proteins (YTHDF 1-3, YTHDC1/2 and IGF2BP 1-3) are mainly responsible for identifying methylation sites and performing dynamic regulation and are closely related to gene expression regulation. Studies have shown that m6A may be involved in biological processes such as mRNA transcription, selective cleavage, nuclear transport, translation, and degradation, resulting in RNA dysfunction, which in turn affects a range of animal vital activities.
At present, research on YTHDF1 protein functions is mainly focused on aspects of tumorigenesis, organism development and the like, but the detailed action mechanism and the biological functions thereof in retina are not clear, so that the development and application of the YTHDF1 protein are limited. Therefore, the intensive research on the treatment and etiology of the retinal pigment degeneration disease by YTHDF1 has great potential.
The invention is realized in the following way:
the invention provides a construction method of a retinal pigment degeneration disease model, which comprises the following steps: the Ythdf1 gene sequence in the genome of the target animal is not expressed or is inhibited.
The inventors have found for the first time that a model of a retinal pigment-modified disease exhibiting characteristics associated with the retinal pigment-modified disease can be obtained such that the Ythdf1 gene sequence in the genome of the target animal is not expressed or is inhibited. Such as rod cell death, is mainly manifested by photoreceptor damage, degeneration, progressive thinning of the outer retinal nuclear layer until disappearance, and corresponding pathological changes in the outer retinal network and other relevant cell layers. Therefore, the novel model of the retinal pigment degeneration disease can be used in the fields of research of the retinal pigment degeneration disease and the like, and provides a richer model selection for research of the disease such as pathogenesis, mechanism and screening of related drugs. In addition, the development of the disease model provides a new target for the treatment or prevention of RP diseases.
In a preferred embodiment of the present invention, the above construction method comprises modification by one or a combination of mutation, deletion and insertion such that the Ythdf1 gene sequence in the genome of the target animal is not expressed or is inhibited.
In one embodiment, the mutation modification means that the corresponding protein site of the Ythdf1 gene is subjected to amino acid change by the corresponding mutation to realize that the protein expressed by the Ythdf1 gene does not have a normal bioactive function or translation is terminated prematurely.
When a deletion modification is employed, it may be a deletion of one or more nucleotides, thereby rendering the Ythdf1 gene sequence non-expressed or less active. For example, deletion modification of the target gene may be achieved by deleting a part or all of the exons.
When the modification is adopted, at least one nucleotide is inserted into the Ythdf1 gene, for example, one or more nucleotides are inserted into an exon to cause frame shift mutation, so that the primary structure, the secondary structure or the tertiary structure of the expressed protein is changed, and the protein expressed by the Ythdf1 gene does not have normal biological activity function.
Therefore, whatever modification is selected, for example, combination modification, is within the scope of the present invention as long as the Ythdf1 gene sequence in the genome of the target animal is not expressed or is inhibited so that the target animal has the characteristics associated with the retinitis pigmentosa disease.
In a preferred embodiment of the present invention, the full-length sequence or partial sequence of the Ythdf1 gene in the genome of the target animal is not expressed or is inhibited by modification by one or a combination of mutation, deletion and insertion.
For example, such that the full length sequence, cDNA sequence or part of the exon sequence of the Ythdf1 gene is not expressed or is inhibited.
In a preferred embodiment of the present invention, the modification is performed by one or a combination of mutation, deletion and insertion such that the exon sequence or a part of the exon sequence of the Ythdf1 gene in the genome of the target animal is not expressed or is inhibited.
In a preferred embodiment of the present invention, at least one of the 1 st to 5 th exons of the Ythdf1 gene in the genome of the target animal is not expressed or is inhibited from being expressed by one or a combination of several of mutation, deletion and insertion.
In one embodiment, the 1 st, 1 st and 3 rd exons, 3 rd and 5 th exons sequences, 3 rd to 5 th exons sequences of the Ythdf1 gene are not expressed or are inhibited from expression.
In a preferred embodiment of the present invention, the 3 rd exon of the Ythdf1 gene in the genome of the target animal is not expressed or is inhibited by one or a combination of several of mutation, deletion and insertion.
In a preferred embodiment of the invention, the above construction method comprises modification of mutation, deletion or insertion by a combination of one or more of the following techniques:
gene knockout technology and gene editing technology.
In a preferred embodiment of the present invention, the gene knockout technology is Cre-loxP gene knockout technology.
In a preferred embodiment of the application of the present invention, the above-mentioned gene editing technology is selected from any one or a combination of several of CRISPR/Cas9 technology, ZFN technology and TALEN technology. It should be noted that, based on other gene editing techniques, it is within the scope of the present invention to make the target animal show the characteristics of the retinitis pigmentosa disease.
In a preferred embodiment of the application of the present invention, the target animal is a non-human mammal. In other embodiments, the non-human mammal is selected from any one of a mouse, a rat, a dog, a pig, a monkey, and an ape. One skilled in the art can adaptively select any non-human mammal as a target animal according to needs, and all the non-human mammals belong to the protection scope of the invention.
The target animal according to the present invention is not limited to the above-described animal, and may be any other type of animal such as rabbit, cow, horse, sheep, etc. Any animal can be selected as long as it is an animal having the Ythdf1 gene, and the Ythdf1 gene is knocked out from a rod cell of the animal to exhibit characteristics of a retinitis pigmentosa disease, and the animal is used as a retinitis pigmentosa disease model in the field of retinitis pigmentosa disease research, and belongs to the scope of the present invention.
In a preferred embodiment of the present invention, the above construction method is directed to the Ythdf1 gene in the genome of all cells of the target animal.
Further, in some embodiments of the present invention, the method for constructing a disease model specifically includes the following steps:
(1) Synthesis of a gRNA sequence SEQ ID NO:1 for replacing exon 3 of the Ythdf1 gene to be knocked out;
(2) The donor vector of the gRNA of the mouse Ythdf1 gene obtained in the step (1) and Cas9 mRNA are subjected to high-flux electrotransformation to obtain a mouse embryo stem cell with the Ythdf1 gene subjected to conditional knockout;
(3) Transplanting the fertilized eggs obtained in the step (2) into the uterus of a pseudopregnant mouse, delivering a offspring mouse, and sequencing and identifying to obtain a first-established mouse with Ythdf1 gene knocked out;
(4) Mating and breeding the chimeric mice obtained in the step (3) and wild mice, and screening heterozygote mice knocked out by Ythdf1 genes in offspring;
(5) Mating and breeding Ythdf1 gene knockout heterozygote mice obtained in the step (4) to obtain Ythdf1 gene knockout homozygote mice, and using the mice as a retinal pigment degeneration disease model.
The animal model obtained by the construction method has typical characteristics of the retinal pigment degeneration disease, has very wide application prospect, and can be used for researching the pathogenesis of the retinal pigment degeneration disease, for example, the animal model provides a basis for deeply knowing and researching the retinal pigment degeneration disease. Or it can be used for screening drugs for preventing or treating retinal pigment-modified diseases, evaluating the efficacy or prognosis of drugs, etc.
The application of the retinal pigment degeneration disease model obtained by the construction method in screening medicines for preventing or treating retinal pigment degeneration diseases.
In a preferred embodiment of the application of the present invention, the application includes: administering a candidate drug to the model of a retinal pigment-modified disease, wherein the candidate drug is indicated as a drug for preventing or treating the retinal pigment-modified disease if the model of the retinal pigment-modified disease undergoes at least one of the following changes before and after administration of the candidate drug:
(1) After administration of the candidate drug, vision in the model of the retinitis pigmentosa disease is improved compared to before administration of the candidate drug; preferably a darkness vision improvement;
(2) After administration of the candidate drug, the outer retinal nuclear layer or inner retinal nuclear layer of the model of the retinitis pigmentosa disease is thickened compared to before administration of the candidate drug;
(3) Following administration of the candidate drug, the epiretinal node of the model of the retinitis pigmentosa disease increases as compared to prior to administration of the candidate drug.
The invention has the following beneficial effects:
the invention discovers for the first time that the Ythdf1 gene sequence in the genome of the target animal is not expressed or is inhibited to obtain a model of the retinal pigment degeneration disease, and the model shows the relevant characteristics of the retinal pigment degeneration disease. Such as rod cell death, is mainly manifested by photoreceptor damage, degeneration, progressive thinning of the outer retinal nuclear layer until disappearance, and corresponding pathological changes in the outer retinal network and other relevant cell layers. Therefore, the novel model of the retinal pigment degeneration disease can be used in the fields of research of the retinal pigment degeneration disease and the like, and provides a richer model selection for research of the disease such as pathogenesis, mechanism and screening of related drugs. In addition, the development of the disease model provides a new target for the treatment or prevention of RP diseases.
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 therefore 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 the construction and identification of a mouse with a specific knockout of Ythdf1 gene (KO) from a retinal rod cell;
fig. 2: detecting Ythdf1 gene knockout efficiency;
fig. 3: electroretinogram (ERG) detection results;
fig. 4: specific knockout of retinal rod cells Ythdf1 gene mouse retinal section immunohistochemical staining results;
fig. 5: specific knockout of retinal rod cells Ythdf1 gene mouse IHC staining result 1;
fig. 6: specific knock-out of retinal rod cells Ythdf1 gene mice IHC staining results 2.
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 embodiment provides a construction method of a retinal pigment degeneration disease model, which takes a mouse as a target animal.
Ythdf1 gene knockout is mediated by Cre/loxP recombinase system, the knockout route is shown in FIG. 1A, and the specific operation is as follows:
1) Ythdf1 knockout mice were purchased from Siro Biotech Inc., taiku Chamber, jiangsu, su, taku, sha Xizhen Zhen Jixi Lu 69, (https:// www.cyagen.com/cn/zh-cn/sperm-bank-live/228994), and exon 3 of the model was deleted (FIG. 1A).
2) Mating and breeding the chimeric mice obtained in the step 1) and wild mice, and screening heterozygote mice knocked out by Ythdf1 genes from offspring;
3) Mating and breeding the Ythdf1 gene knockout heterozygote mice obtained in the step 2) to obtain Ythdf1 gene knockout homozygote mice, and the Ythdf1 gene knockout homozygote mice can be used as a retinal pigment degeneration disease model.
In this example, the offspring mice obtained in the above steps are subjected to genotyping, and the method is as follows:
1) Cutting a small amount of tissue samples from the tail tips of the mice, and placing the small tissue samples into a clean 1.5ml centrifuge tube;
2) 100 μl of lysate (40mM NaOH,0.2mM EDTA solution) was added to the centrifuge tube and heated at 100deg.C in the metal bath for 1h;
3) The tube was removed, cooled to room temperature, 100. Mu.l of a neutralization solution (40 mM Tris-HCl, pH 5.5) was added, and 10000g of the tube was centrifuged for 2 minutes, and the supernatant was used for genotyping of mice.
4) And (3) PCR amplification: the PCR reaction system was configured as follows:
2×Taq Mix 10μL
tail tissue lysate 2. Mu.L
Primer 1 (Ythdf 1-loxP-Forward 1), 1. Mu.L (concentration: 10 mM)
Primer 2 (Ythdf 1-loxP-Forward 2), 1. Mu.L (concentration: 10 mM)
Primer 3 (Ythdf 1-loxP-Reverse), 1. Mu.L (concentration: 10 mM)
ddH 2 O 4μL。
The amplification primer sequences were as follows:
ythdf1-Forward1 sequence:
5’-CTGACTGGTCCTCTGTTGCTAGG-3’；
ythdf1-Forward2 sequence:
5’-CATGGAGATTGGGTACAGGCAGAC-3’；
ythdf1-Revers sequence:
5’-GTCCAAGAGACCCTGCATCACTG-3’；
the amplification procedure was as follows:
after the PCR reaction system is prepared, the template DNA is fully denatured by preheating for 5 minutes at 95 ℃ on a PCR instrument, and then the amplification cycle is carried out. In each cycle, the template is denatured prior to maintaining at 95 ℃ for 30 seconds, then the temperature is reduced to the renaturation temperature of 60 ℃ for 30 seconds, and the primer and the template are fully annealed; the reaction was kept at 72℃for 45 seconds, and primers were allowed to extend on the template to synthesize DNA, thereby completing one cycle. This cycle was repeated 25 times to accumulate a large amount of amplified DNA fragments. Finally, the product was left to extend intact at 72℃for 5 minutes and stored at 4 ℃.
5) Gel electrophoresis
1g of agarose was weighed and placed in 100ml of TAE buffer, and melted in a microwave oven to prepare 1% agarose gel. 10ul of PCR product was taken in the wells and subjected to 120V constant pressure agarose electrophoresis for 15min.
FIG. 1B shows the results of Ythdf1 knockout mice, WT shows a wild-type control, and the band size is 1569bp; het represents a heterozygote with two bands 1569bp and 792bp; KO represents homozygote, and the band size is 792bp. According to the results shown in FIG. 1B, the PCR identification method provided in this example can effectively identify the genotype (heterozygous and homozygous) of the newborn mice for subsequent study.
Example 2
In this example, the gene knockout efficiency in the retinas of Ythdf1 knockout mice was analyzed by immunoblotting (Western blot) experiments.
The method comprises the following steps:
1) The control (Ythdf 1) +/+ ) And Ythdf1 knockout homozygote mice (Ythdf 1) -/- ) After sufficiently grinding, 200ul of protein lysate RIPA (Soy Biotechnology Co., ltd.) was added.
2) After sonicating the cells, they were lysed on ice for 20min.
3) After centrifugation at 16000g for 10min at 4℃the supernatant was transferred to another clean centrifuge tube, 50. Mu.l of protein loading solution was added and mixed well and heated at 95℃for 5min.
4) After the sample was cooled, 20. Mu.l of each sample was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) at 160V to separate proteins.
5) After SDS-PAGE is finished, cutting a nitrocellulose membrane with proper size according to the requirement, paving filter paper, glue, the nitrocellulose membrane and the filter paper in sequence, removing bubbles, putting the membrane transferring groove into an ice-water bath, and transferring the membrane by adopting a constant current of 0.28A for 2 hours.
6) After the transfer of the film is finished, the nitrocellulose film is washed once by pure water, dried and marked. Then blocked with 8% skim milk for 2h.
7) After blocking was completed, a certain amount of primary antibody diluted in blocking solution in a certain proportion (according to the instructions for antibody use) was added and incubated overnight at 4 ℃.
8) The primary antibody was recovered, washed with 1 XTBE buffer 4 times for 10min each, and appropriate secondary antibody was selected depending on the source of primary antibody, and horseradish peroxidase (HRP) -labeled secondary antibody was diluted with 1 XTBE and incubated on a shaker at room temperature for 2h.
9) After the secondary antibody incubation was completed, the membrane was washed 3 times with 1 XTBST for 10min each, and the protein was detected with Thermo ELC luminescence kit using a Bio-Rad chemiluminescent gel imaging system.
The results are shown in FIG. 2. At 3 months of age, ytdf 1 was no longer expressed in the retinal cells of the knockout mice, YTHDF1 protein disappeared in retinal expression, indicating that it had been knocked out, and also demonstrated that example 1 successfully constructed Ytdf 1 knockout homozygote mice (Ytdf 1 -/ -)。
Example 3
This example shows that the Ythdf1 gene knockout homozygote mice constructed in example 1 at 8 months of age (Ythdf 1 -/- ) Performing ERG vision test:
1) Dark adaptation animals should adapt overnight, and the environment should be absolutely clear;
2) The next day of anesthesia: weighing and injecting into abdominal cavity; deep anesthesia is preferred;
3) Animal fixation and mydriasis: after anesthesia was completed, mice were fixed with tape in front of animal test platform under dark red light: the mice are required to be ensured to lie on the ground, namely, the eyes are consistent in height relative to the stimulation port of the flash stimulator, and are fully exposed, and mydriatic agent is dripped.
4) Electrode installation: preheating a retinogram instrument (Espion Visual Electrophysiology System, diagnosyllc, lt-tleton, MA, USA), coating conductive paste on the electrode, clamping the tail of a mouse, and inserting the mouse into an amplifier ground interface; the double-ended needle electrode is inserted into the back cervical skin (approximately in the middle of two ears) and is simultaneously connected with the negative interfaces of the two channels; the gold ring electrode was clamped on the electrode holder of the animal experiment platform, the angle was carefully adjusted, and the center tip of the cornea was slightly contacted. One channel positive electrode is connected with the right eye, and the two channel positive electrodes are connected with the left eye. The contact effect of the gold ring electrode and cornea is improved by dropping normal saline to eyes through the needle tube. The two gold ring electrodes are ensured to contact the same position of the center positive end of the cornea of two eyes at the same angle and in the same mode.
5) Record displayAfter the wave signal is confirmed to be correct, the dark red light is turned off. It can be tried to record the dark adaptation light intensity of 0.003 cd/s.m 2 And (2) confirming the quality of the following signal: if the amplitude of the eyes is greatly different from that expected, it is recommended to check the mounting position of the gold ring electrode again. Then sequentially recording dark adaptation light intensity of 3.0/10.0 cd/s.m 2 After recording the system will automatically turn on the backlight.
6) Continuously recording the light adaptation light intensity of 10.0 cd/s.m 2 Is a signal of (a).
The results found that at 8 months, both a-wave and b-wave of the knockout mice were significantly reduced under dark adaptation conditions compared to wild-type mice, indicating that Ythdf1 resulted in impaired vision after rod cell knockout (fig. 3). FIG. 3A shows the electroretinogram waveforms of Ythdf1 wild type mice in dark adaptation and light adaptation at different light intensities; b is the electroretinogram waveform of Ythdf1 knockout mice under dark adaptation and light adaptation under different light intensities; C-E is a-wave and b-wave statistics for dark adaptation 3.0 and 10.0 and light adaptation 10.0, respectively, indicating that knockout mice have significantly reduced scotopic vision. The electroretinogram a wave and b wave have no obvious difference under the light condition, and the Ythdf1 does not influence the functions of the cone cells.
Example 4
This example the retinas of the knockout mouse model constructed in example 1 were subjected to paraffin section H & E staining.
Retinas of 8 month old mice were stained by paraffin section, hematoxylin-eosin staining (H & E staining method) as follows:
1) Quickly taking eyeball tissues of a mouse, and placing the eyeball tissues in a fixing solution for fixing for 24 hours;
2) Embedding paraffin, slicing with thickness of 4 μm;
3) Slices were conventionally dewaxed with xylene, washed with multi-stage ethanol to water: xylene (I) 5 min- & gt xylene (II) 5 min- & gt 100% ethanol 2 min- & gt 95% ethanol 1 min- & gt 80% ethanol 1 min- & gt 75% ethanol 1 min- & gt distilled water washing 2min;
4) Hematoxylin staining for 5 minutes, washing with tap water;
5) Ethanol hydrochloride differentiation for 30 seconds;
6) Soaking in tap water for 15 minutes;
7) And (5) placing eosin solution for 2 minutes.
8) Conventional dehydration, transparency and sealing sheet: 95% ethanol (I) 1min, 95% ethanol (II) 1min, 100% ethanol (I) 1min, 100% ethanol (II) 1min, xylenol carbonic acid (3:1) 1min, xylene (I) 1min, xylene (II) 1min and neutral resin sealing.
9) And photographing under a microscope.
As a result, it was found that at 8 months, compared with Ythdf1 +/+ (wild) mice, ythdf1 -/- The thickness of the outer and inner layers of the visual network was significantly thinner in (knockout) mice, indicating photoreceptor cell death (fig. 4). FIG. 4A shows Ythdf1 gene mouse retinal paraffin section H&E staining results, FIG. 4B is a graph showing statistics of thickness of outer nuclear layer of retina of Ythdf1 knockout mice at different sites, black line Ythdf1 +/+ (wild) mice, the red line (i.e., the lower line in FIG. 4B) is Ythdf1 -/- (knockout) mice.
Example 5
The present example performs an immunostaining experiment on frozen sections of retina.
The method specifically comprises the following steps: after the Ythdf1 gene knockout mice constructed in example 1 of 8 months old were sacrificed at the neck, eyeballs were quickly taken and put into 4% PFA, fixed on ice for 15min, cut on cornea, and then fixation on ice was continued. After 2h, PBS buffer was washed 3 times, then the eyeball was dehydrated in 30% sucrose solution for 2h, then the cornea and crystals were cut off under a dissecting scope, OCT was embedded and rapidly frozen in a refrigerator at-80 ℃. After about 10min, the OCT embedded eyeball is taken out, and the eyeball is placed in a frozen microtome for balancing at the temperature of minus 25 ℃ for about 30min, and then the eyeball can be sectioned. The slice thickness was 12. Mu.m.
After slicing, higher quality pieces were selected and placed in an oven at 37 ℃ for 30min, then an immunohistochemical pen was circled around the area with retinal tissue, washed three times with PBS to remove OCT, then 5% NDS (containing 0.25% triton) were blocked through for 2h, primary antibodies were incubated, and overnight at 4 ℃. The following day, after three times of PBS washing, the corresponding fluorescent secondary antibodies are incubated, and then three times of PBS washing are carried out, and the plates are sealed and observed.
The results are shown in the graphShown at 5. At 8 months of age, the outer node antibodies Rhodopsin, PDE6B and CNGA1 were stained by frozen tissue sections of the retina, as compared to Ythdf1 +/+ (wild) mice, ythdf1 -/- The outer segments of the retinas of (knockdown) mice were significantly shortened and a significant degeneration characterization occurred.
In FIG. 5A, the inner and outer segments of rod cells are labeled with Rhodopsin and NaK antibodies, respectively, and the nuclei are counterstained with DAPI; FIG. 5B shows the counterstaining of nuclei with DAPI by labeling rod cell light transduction related proteins with PDE6B and CNGA1 antibodies, respectively.
Example 6
This example provides an immunostaining experiment on frozen sections of retina, with mice of 5 months of age in this application compared to example 5.
After the Ythdf1 gene knockout mice constructed in example 1 of 8 months old were sacrificed at the neck, eyeballs were quickly taken and put into 4% PFA, fixed on ice for 15min, cut on cornea, and then fixation on ice was continued. After 2h, PBS buffer was washed 3 times, then the eyeball was dehydrated in 30% sucrose solution for 2h, then the cornea and crystals were cut off under a dissecting scope, OCT was embedded and rapidly frozen in a refrigerator at-80 ℃. After about 10min, the OCT embedded eyeball is taken out, and the eyeball is placed in a frozen microtome for balancing at the temperature of minus 25 ℃ for about 30min, and then the eyeball can be sectioned. The slice thickness was 12. Mu.m.
After slicing, higher quality pieces were selected and placed in an oven at 37 ℃ for 30min, then an immunohistochemical pen was circled around the area with retinal tissue, washed three times with PBS to remove OCT, then 5% NDS (containing 0.25% triton) were blocked through for 2h, primary antibodies were incubated, and overnight at 4 ℃. The following day, after three times of PBS washing, the corresponding fluorescent secondary antibodies are incubated, and then three times of PBS washing are carried out, and the plates are sealed and observed.
The results are shown in FIG. 6. At 5 months of age, the neuroglia marker GFAP is stained by frozen tissue sections of the retina, and compared with a wild type mouse, the retina of the knockout mouse shows obvious gliosis and enhanced inflammatory response, which indicates retinal damage.
In summary, it can be seen that, in the embodiment of the invention, by taking a mouse as an example, the CRISPER/Cas9 knockout technology is used for knocking out the Ythdf1 gene, so that the mouse shows typical characterization of retinal pigment degeneration diseases such as visual impairment, shortening and degradation of extraocular nodes, loss of visual rod cells and the like. Thus, it is fully demonstrated that the knockout of the Ythdf1 gene can cause the target animal to exhibit retinal pigment degeneration disease. The Ythdf1 knockout animal can be used as a model of retinal pigment degeneration disease. The disease model can be used in the fields of research of retinal pigment degeneration diseases and the like, and provides a new model for research of the diseases such as pathogenesis, mechanism and screening of related medicines.
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.
Claims (5)
1. The construction method of the retinal pigment degeneration disease model is characterized by comprising the following steps: modification by deletion allows for the genome of all cells of a mouseYthdf1Exon 3 of the gene was not expressed to give Ythdf1 knockout homozygote mice.
2. The construction method according to claim 1, characterized in that it comprises the deletion modification by means of a combination of one or several of the following techniques:
gene knockout technology and gene editing technology; the gene knockout technology is Cre-loxP gene knockout technology, and the gene editing technology is selected from any one or a combination of a plurality of CRISPR/Cas9 technology, ZFN technology and TALEN technology.
3. Use of a model of a retinitis pigmentosa disease obtained by the construction method according to any one of claims 1-2 for screening a medicament for preventing or treating a retinitis pigmentosa disease.
4. The application according to claim 3, characterized in that it comprises: administering a candidate drug to the model of a retinal pigment-modified disease, wherein the candidate drug is indicated as a drug for preventing or treating a retinal pigment-modified disease if the model of a retinal pigment-modified disease changes at least one of the following before and after administration of the candidate drug:
(1) An improvement in vision of the model of the retinal pigment degeneration disease after administration of the drug candidate compared to before administration of the drug candidate;
(2) After administration of the candidate drug, the outer retinal nuclear layer or inner retinal layer of the model of the retinitis pigmentosa disease is thickened compared to before administration of the candidate drug;
(3) After administration of the candidate drug, the epiretinal node of the model of the retinitis pigmentosa disease increases as compared to before administration of the candidate drug.
5. The use according to claim 4, wherein the vision improvement of the model of retinitis pigmentosa disease is: the darkness vision of the model of the retinal pigment degeneration disease is improved.
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