CN114916502A - Construction method and application of retinal pigment degeneration disease model - Google Patents
Construction method and application of retinal pigment degeneration disease model Download PDFInfo
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- CN114916502A CN114916502A CN202210802107.2A CN202210802107A CN114916502A CN 114916502 A CN114916502 A CN 114916502A CN 202210802107 A CN202210802107 A CN 202210802107A CN 114916502 A CN114916502 A CN 114916502A
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Abstract
The invention discloses a construction method and application of a retinal pigment degeneration disease model, and relates to the technical field of medical engineering. It includes: knocking out Ythdc2 gene sequence in non-human target mammal retina rod cell genome to obtain retina pigment degeneration disease model. The disease model exhibits characteristics associated with retinitis pigmentosa disease. For example, rod cell death is mainly manifested by photoreceptor damage and degeneration, gradual thinning and disappearance of the outer nuclear layer of the retina, and corresponding pathological changes of the outer retinal layer and other related cell layers. Therefore, the animal with the knockout of the Ythdc2 gene in the retinal rod cells can be used as a retinal pigment degeneration disease model, is used in the fields of research of retinal pigment degeneration diseases and the like, and provides a new model for the research of the diseases, such as the pathogenesis, the mechanism and the screening of related preventive or therapeutic drugs.
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 (RP) is a progressive, inherited, dystrophic retinal degeneration. RP, a hereditary blinding ocular fundus disease, can be divided into two categories based on clinical phenotype: typical RP patients exhibit widespread impairment of rod cells, accounting for approximately 80% -90% of RP patients; whereas atypical RP, which accounts for only 10% -20% of RP patients, is mainly cone cell damage. The typical RP patients first develop night blindness and progressive visual field impairment due to rod cell dysfunction, progressing to tubular fields, until blindness. Pathologically, classical RP primarily affects rod cells, causing rod cell death and subsequent cone cell death, manifested by photoreceptor damage, degeneration, progressive thinning of the outer nuclear layer of the retina until disappearance, with corresponding pathological changes in other relevant cell layers of the retina.
The incidence rate of RP in Chinese population is 1/3500. At present, diagnosis and treatment of RP still face many difficulties, which are mainly due to high heterogeneity of clinical phenotype and heredity, and lack of systematic research on its pathological mechanism, resulting in unclear specific molecular mechanism of RP, which brings great hindrance to clinical diagnosis and treatment of RP, so it is necessary to develop new disease model and deeply research on detailed pathogenic mechanism of RP. At present, however, there is a lack of corresponding RP disease models.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a construction method of a retinal pigment degeneration disease model and application thereof to solve the technical problems.
The invention is realized in the following way:
the invention provides a method for constructing a retinal pigment degeneration disease model, which comprises the following steps: knocking out Ythdc2 gene sequence in non-human target mammal retina rod cell genome.
YTHDC2 (YTHDomalnContaining 2, MGI:2448561), which is located on mouse chromosome 18, 44961521-45022787bp, 61.26kb in total length, and 6299bp in total cDNA length, comprising 30 exons.
YTHDC2 as the RNA m6A methylation reading protein, recognition of RNA methylation sites and further regulation, it is widely distributed in various types of tissue cells. m6A methylation is a dynamic reversible process, the formation process is mainly composed of methyltransferase complex (METTL3, METTL14 and WTAP, etc.), the demethylation process is completed by demethylase (FTO and ALKBH5), and the reading protein (YTHDF1-3, YTHDC1/2, IGF2BP1-3, etc.) is mainly responsible for recognizing methylation site and dynamically regulating, and is closely related to gene expression regulation. Secondly, m6A may be involved in biological processes such as mRNA transcription, selective splicing, nuclear transport, translation and degradation, thereby causing RNA dysfunction and further influencing a series of animal life activities. At present, the functions of YTHDC2 protein are increasingly researched, including the influence on tumorigenesis, body development and the like, but the detailed action mechanism and the biological function of YTHDC2 protein in retina are not clear, so that the development and application of YTHDC2 protein are limited. Therefore, extensive research on the treatment and the cause of the retinal pigment degeneration disease by YTHDC2 has great potential.
The inventor researches and discovers that the Ythdc2 gene in the target animal rod cells is knocked out, so that the target animal can show the characteristics related to the retinitis pigmentosa disease. For example, rod cell death is mainly manifested by photoreceptor damage and degeneration, gradual thinning and disappearance of the outer nuclear layer of the retina, and corresponding pathological changes of the outer retinal layer and other related cell layers. Therefore, the animal with the knockout of the Ythdc2 gene in the retinal rod cells can be used as a retinal pigment degeneration disease model, is used in the fields of research of retinal pigment degeneration diseases and the like, and provides a new model for the research of the diseases, such as the pathogenesis, the mechanism and the screening of related medicines.
The construction of the disease model is beneficial to providing a new target for the treatment or prevention of the retinitis pigmentosa disease.
The inventor researches to obtain: the YTHDC2 protein has important functions in retina, can recognize the methylation site of mRNA to regulate the expression of genes, thereby influencing the function of retina, directly or indirectly influencing the survival of photoreceptor cells, and causing retinitis pigmentosa. However, the specific pathogenic molecular mechanism is not clear and is worth further research.
The sequences of the above-described knockout of the Ythdc2 gene in the retinal rod cell genome include, but are not limited to: full-length sequence and partial sequence of Ythdc2 gene. The Ythdc2 gene is knocked out, and the Ythdc2 gene sequence is only required to silence the expression of Ythdc2 gene in a rod cell, so that a non-human target mammal can show characteristics related to retinal pigment degeneration diseases, and the invention belongs to the protection scope of the invention. For example, rod cell death is mainly manifested by photoreceptor damage and degeneration, gradual thinning and disappearance of the outer nuclear layer of the retina, and corresponding pathological changes of the outer retinal layer and other related cell layers.
In a preferred embodiment of the invention, the exon sequence of the Ythdc2 gene in the genome of the retinal rod of the non-human target mammal is knocked out. Including without limitation any one or more of exon 1 through exon 30 of the knockout Ythdc2 gene.
In a preferred embodiment of the invention, the No. 3 exon sequence of the Ythdc2 gene is knocked out; or knocking out exon 3 of Ythdc2 gene and knocking out at least one of exons 1-2 and 4-30.
The inventors have found that, by knocking out exon 3 sequence of the Ythdc2 gene, a non-human target mammal shows characteristics related to retinal pigment degeneration diseases, such as rod cell death, mainly manifested by photoreceptor damage and degeneration, gradual thinning and disappearance of the outer nuclear layer of the retina, and corresponding pathological changes of the outer retinal layer and other related cell layers.
In other embodiments, exon 3 and at least one other exon sequence of the Ythdc2 gene may also be knocked out simultaneously or separately to obtain a non-human target mammal having characteristics associated with retinitis pigmentosa disease.
For example, the 3 rd exon and the 4 th exon sequences of the Ythdc2 gene are knocked out simultaneously, and the 3 rd exon and the 2 nd exon sequences of the Ythdc2 gene are knocked out simultaneously.
In a preferred embodiment of the present invention, the gene editing technique used for the knockout is selected from: at least one of CRISPR/Cas9 technology, artificial nuclease-mediated Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Cre-loxp gene knockout technology.
In a preferred embodiment of the application of the present invention, the gene editing technology adopted by the knockout is selected from Cre-loxp gene knockout technology and CRISPR/Cas9 technology.
In a preferred embodiment of the present invention, the construction method comprises:
mutually mating and breeding the Ythdc2 gene Flox heterozygote non-human target mammals to obtain Ythdc2 gene Flox homozygote non-human target mammals;
then mating the Ythdc2 gene Flox homozygote non-human target mammal with the Rod-Cre gene-transferred non-human target mammal to obtain the retina Rod cell knockout Ythdc2 gene non-human target mammal.
In a preferred embodiment of the use of the invention, the non-human target mammal is selected from any one of a mouse, rat, horse, cow, sheep, rabbit, dog, pig, monkey, ape and orangutan.
The target animal of the present invention is not limited to the above-mentioned animals. Whatever animal is selected, the animal with the Ythdc2 gene can be used as a target animal in the construction method of the invention, the Ythdc2 gene is knocked out in a rod cell of the animal, so that the animal shows the characteristic of the retinal pigment degeneration disease, and the animal is used as a retinal pigment degeneration disease model in the research field of the retinal pigment degeneration disease and belongs to the protection scope of the invention.
In a preferred embodiment of the use of the invention, the non-human target mammal is selected from the group consisting of mice. For example, a Ythdc2 gene Flox mouse (C57BL/6-Ythdc2em1(Flox) Smoc, available from Shanghai, Square model Biotech, Inc.) in which a loxP site is inserted upstream and downstream of exon 3 of Ythdc2 gene; the obtained Ythdc2 gene Flox heterozygote mice are mutually crossed and bred to obtain Ythdc2 gene Flox homozygote mice (Ythdc 2) flox/flox ) (ii) a Then the Ythdc2 gene Flox homozygote mouse and Rod-Cre gene transfer mouse (B6.Cg-Pde6 b) + Tg (Rho-icre)1Ck/Boc, purchased from Jackson laboratory, USA, MGI:4417915), mating, and Rod drives Cre gene to be specifically expressed in retinal Rod cells, so as to obtain a retinal Rod cell knockout Ythdc2 gene mouse, and after identification, the mouse with correct identification result is used as a retinal pigment degeneration disease model.
The retinal pigment degeneration disease model constructed by the construction method of the retinal pigment degeneration disease model is applied to screening of drugs for preventing or treating the retinal pigment degeneration disease.
If the candidate drug is administered, vision in the retinitis pigmentosa disease model is improved as compared to before administration of the candidate drug; it indicates that the candidate drug may be a drug for the prophylaxis or treatment of retinal pigment degeneration diseases.
If the drug candidate is administered, the outer retinal cortex tends to be thicker or have a tendency to thicken compared to before the drug candidate is administered; it indicates that the candidate drug may be a drug for the prophylaxis or treatment of retinal pigment degeneration diseases.
If the candidate drug is administered, the extraretinal segment grows and the degenerative characteristic weakens or disappears as compared to before the candidate drug is administered; it indicates that the candidate drug may be a drug for the prophylaxis or treatment of retinal pigment degeneration diseases.
The application of the retinal pigment degeneration disease model constructed by the construction method of the retinal pigment degeneration disease model in the research of retinal pigment degeneration diseases aims at the diagnosis or treatment of non-diseases.
The disease model obtained by the construction method has typical characteristics of the retinal pigment degeneration disease, has very wide application prospect, and provides a foundation for deeply understanding and researching the retinal pigment degeneration disease, for example, the disease model is used for researching the pathogenesis and the pathogenesis of the retinal pigment degeneration disease. Or for screening a drug for preventing or treating a retinal pigment degeneration disease, for evaluating the efficacy or prognosis of a drug, or the like.
The invention has the following beneficial effects:
the inventor researches and discovers that: knocking out Ythdc2 gene in target animal rod cell can make target animal show the characteristic related to retinitis pigmentosa disease. For example, rod cell death is mainly manifested by photoreceptor damage and degeneration, gradual thinning and disappearance of the outer nuclear layer of the retina, and corresponding pathological changes of the outer retinal layer and other related cell layers. Therefore, the animal with the knockout of the Ythdc2 gene in the retinal rod cells can be used as a retinal pigment degeneration disease model, is used in the fields of research of retinal pigment degeneration diseases and the like, and provides a new model for the research of the diseases, such as the pathogenesis, the mechanism and the screening of related medicines.
The construction of the disease model is beneficial to providing a new target for the treatment or prevention of the retinitis pigmentosa disease.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.
FIG. 1 shows a retinal Rod cell (Rod) specific knockout Ythdc2 gene mouse (Ythdc 2) RKO ) The glenoid Ythdc2 is selected as a result graph of construction and identification flox/flox The mice were control mice (Ythdc 2) f/f ) (ii) a A: a construction strategy of a Ythdc2 gene knockout mouse, B, a genotype identification result of the Ythdc2 gene knockout mouse;
FIG. 2 is a graph showing the results of the detection of the gene knockout efficiency of Ythdc 2;
FIG. 3 is a graph showing the results of Electroretinogram (ERG) measurements;
FIG. 4 is a diagram showing the immunohistochemical staining result of a retina section of a mouse with a retinal rod cell specific knockout Ythdc2 gene;
FIG. 5 is a graph showing the results of IHC staining in mice with specific knockout of Ythdc2 gene in retinal rods (inner and outer segments of rods labeled with NaK and Rhodopsin antibodies, respectively, and nuclei counterstained with DAPI);
fig. 6 is a graph showing IHC staining results of mice with retinal rod-specific knockout Ythdc2 gene (muller glial cells in the retina were labeled with GFAP antibody, DAPI counterstained nuclei).
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); synthesis of oligonucleotides (oligo Synthesis) (m.j. gate eds., 1984); animal Cell Culture (Animal Cell Culture) (edited by r.i. freshney, 1987); methods in Enzymology (Methods in Enzymology) in Academic Press (Inc.), Handbook of Experimental Immunology in (compiled by D.M.Weir and C.C.Black), Gene Transfer Vectors for Mammalian Cells (compiled by J.M.Miller and M.P.Calos) (compiled by Gene Transfer Vectors in Mammalian Cells, 1987), Methods in Current Molecular Biology (compiled by Current Protocols in Molecular Biology) (compiled by F.M.Ausubel et al, 1987), PCR (compiled by Polymerase Chain Reaction in Polymer Immunology) (compiled by Mullis et al, 1994), and Methods in Current Immunology (compiled by Current Protocols in Molecular Biology, 1987), Methods in PCR (compiled by Current Protocols in Molecular Biology) (compiled by Cologies et al, 1991, each of which is incorporated by Immunology, Inc.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
In this example, a method for constructing a retinal pigment degeneration disease model provided by the present invention is described using a mouse as a target animal, and the route of the Ythdc2 gene knockout is shown in fig. 1 a.
Ythdc2 Gene Flox mice (C57BL/6-Ythdc2em1(Flox) Smoc) were purchased from Hippon, Inc., A model Biotechnology Ltd. loxP sites are inserted upstream and downstream of exon 3 of the mouse Ythdc2 gene.
The obtained Ythdc2 gene Flox heterozygote mice are mutually mated and bred to obtain Ythdc2 gene Flox homozygote mice (Ythdc 2) flox/flox ) (ii) a Then the Ythdc2 gene Flox homozygote mouse and Rod-Cre gene transfer mouse (B6.Cg-Pde6 b) + Tg (Rho-icre)1Ck/Boc, purchased from Jackson laboratory, USA, MGI:4417915), mating, and Rod drives Cre gene to be specifically expressed in retinal Rod cells, so as to obtain a retinal Rod cell knockout Ythdc2 gene mouse (Ythdc 2) RKO )。
Example 2
The offspring mice involved in the construction of the retinitis pigmentosa disease model of example 1 were genotyped as follows:
1) cutting a little tissue sample from the tail tip of the mouse, and placing the cut tissue sample in a clean 1.5ml centrifuge tube;
2) add 100. mu.l lysis buffer (40mM NaOH, 0.2mM EDTA solution) to the centrifuge tube and heat for 1h at 100 ℃ in a metal bath;
3) the tube was removed, cooled to room temperature, 100. mu.l of a neutralizing solution (40mM Tris-HCl, pH5.5) was added thereto, and the mixture was centrifuged at 10000g for 2min, and the supernatant was used for mouse genotyping.
4) And (3) PCR amplification: the PCR reaction system was configured as follows
2×Taq Mix 10μL
Tail tissue lysate 4. mu.L
Primer 1(Ythdc2-loxP-Forward or Rod-Cre-F), 1. mu.L (concentration: 10mM)
Primer 2(Ythdc2-loxP-Reverse or Rod-Cre-R), 1. mu.L (concentration: 10mM)
ddH 2 O 4μL。
The primer sequences are as follows:
ythdc2-loxP-Forward sequence:
5’-GAACAGCCAGCCATCACC-3’；
ythdc2-loxP-Reverse sequence:
5’-AAAAATAGAGCAGTCCAAAGAGTA-3’；
Rod-Cre-F：TCAGTGCCTGGAGTTGCGCTGTGG；
Rod-Cre-R：CTTAAAGGCCAGGGCCTGCTTGGC。
and (3) amplification procedure:
after the PCR reaction system was prepared, the template DNA was fully denatured by preheating at 95 ℃ for 5 minutes in a PCR instrument, and then subjected to an amplification cycle. In each cycle, the temperature is kept for 30 seconds at 95 ℃ to denature the template, then the temperature is reduced to 60 ℃ for renaturation, and the temperature is kept for 30 seconds to fully anneal the primer and the template; the mixture was kept at 72 ℃ for 45 seconds to extend the primer on the template and synthesize DNA, completing one cycle. This cycle was repeated 25 times to allow a large accumulation of amplified DNA fragments. Finally, the product was left intact for 5 minutes at 72 ℃ 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 placed in a well and subjected to 120V constant pressure agarose electrophoresis for 15 min. Imaging with a gel imaging system.
FIG. 1B shows the electrophoresis results, and the top panel of FIG. 1B shows that the upstream and downstream primers of Ythdc2-loxP were used to control wild type mice (Ythdc 2) +/+ ) Heterozygote mice (Ythdc 2) flox/+ (ii) a Rho-Cre) and homozygote mice (Ythdc 2) flox / flox (ii) a Rod-Cre) for PCAnd R amplification detection. FIG. 1B is a graph showing the comparison of upstream and downstream primers of Rod-Cre against wild-type control mice (Ythdc 2) +/+ ) Heterozygote mice (Ythdc 2) flox/+ (ii) a Rho-Cre) and homozygote mouse (Ythdc 2) flox / flox (ii) a Rod-Cre) for PCR amplification detection.
WT represents wild type control, band size 244 bp; het represents a heterozygote, and has two bands of 244bp and 278 bp; KO represents a homozygote and the band size is 278 bp.
From the results of FIG. 1B, it is shown that the employed identification method can effectively identify the genotype of the newborn mouse for subsequent studies.
Example 3
This example analyzes the gene knockout efficiency in the retina of Rod-Cre knockout mice using an immunoblot (Western blot) assay.
The analysis method is as follows:
1) control mice Ythdc2 were obtained separately f/f And knockout mouse Ythdc2 RKO After being sufficiently ground, 200. mu.l of protein lysate RIPA was added to the retina.
2) After cell disruption by sonication, the cells were lysed on ice for 20 min.
3) Centrifuging at 16000g for 10min at 4 deg.C, transferring the supernatant to another clean centrifuge tube, adding 50 μ l protein sample solution, mixing, and heating at 95 deg.C for 5 min.
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 a proper size according to needs, sequentially laying filter paper, glue, the nitrocellulose membrane and the filter paper, removing bubbles, putting a membrane transferring groove into an ice water bath, and transferring the membrane for 2 hours by adopting a constant current of 0.28A.
6) After the membrane is completely transferred, the nitrocellulose membrane is washed by pure water, dried and marked. Then blocked with 8% skimmed milk for 2 h.
7) After blocking was complete, a defined amount of primary antibody diluted in blocking solution in a defined ratio (according to the instructions for antibody use) was added and incubated overnight at 4 ℃.
8) The primary antibody was recovered and the membrane was washed 4 times with 1 × TBST buffer for 10min each time, and depending on the source of the primary antibody, the appropriate secondary antibody was selected, and horseradish catalase (HRP) -labeled secondary antibody was diluted with 1 × TBST and incubated for 2h on a shaker at room temperature.
9) After the secondary antibody incubation was completed, the membrane was washed 3 times with 1 XTSST for 10min each time, and the protein was detected using Thermo's ELC luminescence kit using Bio-Rad's chemiluminescence gel imaging system.
The results are shown in FIG. 2. A-B, analyzing the gene knockout efficiency of Ythdc2 knockout mice retinas by Western blot experiments, and the statistical result shows that Ythdc2 is not expressed any more in knockout mice retinas rod cells when the mice are 2 months old, which indicates that the mice are knocked out.
Example 4
This example shows Ythdc2 in 9-month-old Ythdc2 knockout mice RKO Performing ERG vision detection:
1) dark adaptation animals should adapt dark overnight, and the environment should be absolutely free of light;
2) anesthesia the next day: weighing, and injecting in an abdominal cavity; deep anesthesia is suitable;
3) animal fixation and mydriasis: after anesthesia was completed, mice were taped in front of the animal testing platform under dark red light: the mouse needs to be ensured to lie prone, namely, the height of two eyes is consistent relative to the stimulating port of the flash stimulator, the eyes are fully exposed, and the mydriatic agent is dripped.
4) Electrode installation: preheating an electroretinograph (Espion Visual electrophoresis System, diagnosllc, Lit-tleton, MA, USA), coating conductive paste on the electrodes, clamping the mouse tail, and inserting the electrodes into the "ground" interface of the amplifier; the double-ended needle electrode is inserted into the nape skin (approximately in the middle of the ears) and simultaneously connected with the negative interfaces of the two channels; the gold ring electrode was clamped to the electrode holder of the animal experiment platform, carefully adjusted in angle, and lightly touched the central apex of the cornea. One channel anode is connected with the right eye, and the two channel anodes are connected with the left eye. The needle tube is used for dripping physiological saline on the eyes, so that the contact effect of the gold ring electrode and the cornea is improved. The two gold ring electrodes are ensured to contact the same position of the central positive end of the cornea of the two eyes in the same angle and mode.
5) And after the oscillography signal is recorded to confirm that the oscillography signal is correct, the dark red light is turned off. The dark adaptive light intensity of 0.003 cd/s.m can be recorded by first trying 2 The ERG detection of (a), confirms the quality of the signal: if a large difference from the expected amplitude occurs in the amplitudes of both eyes, it is recommended to check the mounting position of the gold ring electrode again. Then the dark adapted light intensity is recorded as 3.0/10.0 cd/s.m 2 After recording, the system will automatically turn on the backlight.
6) Continuously recording light adaptive light intensity of 3.0 cd/s.m 2 Of the signal of (a).
Results referring to FIG. 3, A, B Ythdc2 wild type and Ythdc2 knockout mice at different light intensities are dark adapted to electroretinogram waveforms; c: dark adaptation 3.0 and 10.0 and light adaptation 10.0, and a wave and b wave statistics, wherein C in FIG. 3 shows that the function of the knockout mouse rod cells is remarkably reduced; d, E, photoperiod adaptation of 3.0 Ythdc2 wild type and Ythdc2 gene knockout mice to electroretinogram waveforms; f: the statistics of a wave, b wave and Flicker wave amplitude of the light adaptation is 3.0, and F in FIG. 3 shows that the function of the visual cone cells of the knockout mice is slightly reduced, but the difference of the a wave and the b wave amplitude has no statistical significance (P < 0.05).
The results of this example show that at 9 months, both a-wave and b-wave were significantly reduced in knockout mice under dark adaptation conditions compared to wild mice, indicating that Ythdc2 caused impaired vision following rod cell knockout.
Example 5
This example paraffin sections the retina and H & E staining. The retinas of 9-month-old mice were subjected to paraffin sectioning and staining by hematoxylin-eosin staining (H & E staining method) as follows:
1) quickly taking eyeball tissues of the mouse, and placing the eyeball tissues in a stationary liquid for fixation for 24 hours;
2) embedding in paraffin, and slicing to obtain slices with a thickness of 4 μm;
3) the slices were dewaxed conventionally with xylene, washed with multi-stage ethanol to water: xylene (I)5min → xylene (II)5min → 100% ethanol 2min → 95% ethanol 1min → 80% ethanol 1min → 75% ethanol 1min → distilled water washing 2 min;
4) hematoxylin staining for 5 minutes and washing with tap water;
5) ethanol hydrochloride is differentiated for 30 seconds;
6) soaking in tap water for 15 minutes;
7) placing in eosin solution for 2 minutes.
8) Conventional dehydration, transparency, mounting: 95% ethanol (I)1min → 95% ethanol (II)1min → 100% ethanol (I)1min → 100% ethanol (II)1min → xylene carbolic acid (3: 1)1min → xylene (I)1min → xylene (II)1min → neutral resin encapsulation.
9) Take pictures under microscope.
The results found that at 9 months, it is comparable to Ythdc2 f/f (control) mouse, Ythdc2 RKO The thickness of the outer nuclear layer of the retina was significantly thinned in (knock out) mice, indicating photoreceptor cell death (figure 4).
In FIG. 4, A: the H & E staining result of the mouse retina paraffin section of the Ythdc2 gene specific knockout mouse retina of the retina rod cells shows that the Outer Nuclear Layer (ONL) and the Inner Nuclear Layer (INL) are thinned.
B in fig. 4: statistics of outer nuclear layer thickness for different distances of the Ythdc2 knockout mouse retina from the Optic Nerve (ON).
Example 6
This example frozen sections the retina and immunostaining: after a 9-month-old mouse with the Ythdc2 gene specifically knocked out is killed by breaking the neck, the eyeball is quickly taken out, the mouse is placed into 4% PFA, the mouse is fixed on ice for 15min, the opening of the cornea is cut, and then the mouse is continuously fixed on ice. After 2h, washing with PBS buffer solution for 3 times, then placing the eyeball in 30% sucrose solution for dehydration for 2h, then cutting off cornea and crystal under a dissecting mirror, embedding with OCT, and rapidly placing in a refrigerator at-80 ℃ for freezing. After about 10min, the OCT embedded eyeball is taken out and placed in a freezing microtome to be balanced at-25 ℃ for about 30min, and then the section can be obtained. The slice thickness was 12 μm.
After slicing, the higher quality slices were selected and placed in an oven at 37 ℃ for 30min, then an immunohistochemical pen was circled in the area with retinal tissue, washed three times with PBS to remove OCT, then blocked with 5% NDS (containing 0.25% Triton) for 2h, incubated with primary antibodies (NaK and Rhodopsin antibodies) overnight at 4 ℃. On the following day, after three washes with PBS, the corresponding fluorescent secondary antibody was incubated, then washed three more times with PBS, mounted, and observed.
The results are shown in FIG. 5. At 9 months of age of the mice, the staining of the internodal and extranodal antibodies Nak and Rhodopsin by retinal frozen tissue sections was compared to Ythdc2 f/f (control) mouse, Ythdc2 RKO The (knockout) mouse epiretinal segment is obviously shortened and has obvious degeneration characterization.
Example 7
Immunostaining of frozen retinal sections: after a 9-month-old mouse with the Ythdc2 gene specifically knocked out is killed by breaking the neck, the eyeball is quickly taken out, the mouse is placed into 4% PFA, the mouse is fixed on ice for 15min, the opening of the cornea is cut, and then the mouse is continuously fixed on ice. After 2h, washing with PBS buffer solution for 3 times, then placing the eyeball in 30% sucrose solution for dehydration for 2h, then cutting off cornea and crystal under a dissecting mirror, embedding with OCT, and rapidly placing in a refrigerator at-80 ℃ for freezing. After about 10min, the OCT embedded eyeball is taken out and placed in a freezing microtome to be balanced at-25 ℃ for about 30min, and then the section can be obtained. The slice thickness was 12 μm.
After slicing, selecting the higher quality slices, placing in an oven at 37 ℃ for 30min, then drawing circles on the places with the retinal tissues by an immunohistochemical pen, washing three times by PBS to remove OCT, then blocking by 5% NDS (containing 0.25% Triton) for permeation for 2h, incubating primary antibody (GFAP antibody), and standing overnight at 4 ℃. The following day, after three washes with PBS, the corresponding fluorescent secondary antibody was incubated, then washed three more times with PBS, mounted, and observed.
The results are shown in FIG. 6. At the age of 9 months of mice, after the Mueller glial cell marker GFAP is sliced by frozen retinal tissues, the retina of a knockout mouse has obvious glial cell hyperplasia and enhanced inflammatory response compared with that of a wild mouse, and therefore, the retina damage is indicated. That is, the constructed animal model shows the characteristics of the retinal pigment degeneration disease.
In conclusion, the embodiment of the invention takes a mouse as an example, and the Ythdc2 gene is specifically knocked out in the retinal rod cells of the mouse by using the CRISPER/Cas9 knock-out technology, so that the mouse shows typical characteristics of retinal pigment degeneration diseases such as impaired vision, short and degenerated outer segments of the visual cells, lost visual cells and the like. It is fully demonstrated that conditional knock-out of the Ythdc2 gene in retinal rods can cause target animals to exhibit retinitis pigmentosa disease. The animal with the Ythdc2 gene conditionally knocked out by the retinal rod cells can be used as a retinal pigment degeneration disease model. The disease model can be used in the fields of research on retinal pigment degeneration diseases and the like, and provides a new model for the research on the diseases, such as the pathogenesis process, mechanism and screening of related medicines.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for constructing a retinal pigment degeneration disease model is characterized by comprising the following steps: knocking out Ythdc2 gene sequence in non-human target mammal retina rod cell genome.
2. The method of claim 1, wherein the exon sequence of the Ythdc2 gene in the genome of the retinal rod of the non-human target mammal is knocked out.
3. The method for constructing according to claim 2, wherein the exon 3 sequence of the Ythdc2 gene is knocked out; or knocking out exon 3 of Ythdc2 gene and knocking out at least one of exons 1-2 and 4-30.
4. The method of construction according to any one of claims 1 to 3, wherein the knockout is performed by a gene editing technique selected from the group consisting of: at least one of CRISPR/Cas9 technology, ZFN technology, TALEN technology and Cre-loxp gene knockout technology.
5. The method of construction according to any one of claims 1-3 wherein the knockout employs a gene editing technique selected from the group consisting of Cre-loxp gene knockout technique and CRISPR/Cas9 technique.
6. The building method according to claim 4, characterized in that the building method comprises:
mating Ythdc2 gene Flox heterozygote non-human target mammals for breeding to obtain Ythdc2 gene Flox homozygote non-human target mammals;
and then mating the Ythdc2 gene Flox homozygote non-human target mammal with a Rod-Cre gene transfer non-human target mammal to obtain a retina Rod cell knockout Ythdc2 gene non-human target mammal.
7. The method according to any one of claims 1 to 6, wherein the non-human target mammal is selected from any one of a mouse, rat, horse, cow, sheep, rabbit, dog, pig, monkey, ape, and orangutan.
8. The method of construction according to any one of claims 1 to 6, wherein the non-human target mammal is selected from the group consisting of mice.
9. Use of the retinal pigment degeneration disease model constructed by the method for constructing a retinal pigment degeneration disease model according to any one of claims 1 to 8 for screening a medicament for preventing or treating a retinal pigment degeneration disease.
10. Use of the retinal pigment degeneration disease model constructed by the method for constructing a retinal pigment degeneration disease model according to any one of claims 1 to 8 for the study of retinal pigment degeneration diseases for the purpose of diagnosis or treatment of non-diseases.
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| CN117296799B (en) * | 2023-11-28 | 2024-02-02 | 四川省医学科学院·四川省人民医院 | Construction method and application of retinal pigment degeneration disease model |
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