CN117230077B - Application of Hakai gene in RP disease model construction and construction method - Google Patents
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Abstract
The invention discloses a method for preparing a composite materialHakaiAn application of gene in RP disease model construction and a construction method thereof relate to the technical field of medical engineering. The construction method is selected from any one of the following methods: (1) Knocking out in the genome of a rod cell of a retina of a non-human target animalHakaiA gene; (2) Gene silencing technology for making retina rod cell genome of non-human target animalHakaiThe expression level of the gene is reduced. By the method, the animal body can be made to show the relevant characteristics of the retinal pigment degeneration disease (RP) disease. The invention provides a new RP disease model for the research of RP diseases and for screening medicaments for treating the diseases, and enriches the model of RP disease research.
Description
Technical Field
The invention relates to the technical field of medical engineering, in particular to a medical engineering machineHakaiApplication of gene in RP disease model construction and construction method.
Background
Currently, retinitis Pigmentosa (RP) is a progressive, inherited blinding fundus disease that leads to vision loss, usually caused by abnormal retinal photoreceptors, clinically manifested as chronic progressive visual field loss, night blindness, pigmentary retinopathy and electroretinogram abnormalities, which ultimately lead to blindness. There is currently a relatively lack of disease models to study RP.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provideHakaiApplication of gene in RP disease model construction and construction method. The invention discovers that the animal body is knocked outHakaiThe gene can cause animals to show related characteristics of RP diseases, and the animals can be used as RP disease models, so that the invention provides a new RP disease model for researching RP diseases and screening medicines for treating the diseases, and enriches the models of RP disease researches.
The invention is realized in the following way:
in a first aspect, the present invention provides a method for constructing a model of a retinal pigment degeneration disease, comprising the method of any one of:
(1) Knocking out in the genome of a rod cell of a retina of a non-human target animalHakaiA gene;
(2) Gene silencing technology for making retina rod cell genome of non-human target animalHakaiThe expression level of the gene is reduced.
HAKAI was originally identified as a ring finger type E3 ubiquitin ligase of the E-cadherin complex, which is the major component of adhesion molecule ligation. HAKAI binds to the cytoplasmic domain of E-cadherin and mediates its ubiquitination, endocytosis, and lysosomal degradation. The absence of E-cadherin leads to disruption of epithelial cell-cell adhesion, a key event in epithelial transformation, and promotes invasion and metastasis. The cytoplasmic domain of E-cadherin is linked to the cytoskeleton by proteins such as β -catenin and α -catenin, which are involved in signaling pathways that regulate cell proliferation, differentiation and survival. After activation of several tyrosine kinases (e.g., EGF or HGF receptors), the E-cadherin complex is phosphorylated at tyrosine residues. Phosphorylation of E-cadherin promotes its recognition by HAKAI, followed by E-cadherin hydrolysis and clearance from cell-cell contact. HAKAI plays a critical role in reducing cell-cell contact in epithelial cells, which is critical to the development and progression of tumors.
HAKAI has been studied mainly in the context of epithelial-mesenchymal transition and cancer progression over the last decades. However, more and more data have found HAKAI to be a component of m6A biogenesis mechanisms in vertebrates and plants. HAKAI was identified as one of the strongest WTAP interacting factors in mammalian cells and was also part of an evolutionarily conserved protein complex including WTAP, VIRMA and ZC3H 1339. Furthermore, HAKAI mutations in arabidopsis exhibit mild developmental defects, while m6A levels are reduced. Recently, HAKAI was identified in the Spenito (Nito) interaction group of drosophila s2r+ cells as one of the most enriched proteins, suggesting an evolutionarily conserved role of HAKAI in the m6A pathway.
HAKAI is expressed in a number of tissues such as heart, brain, liver, retina, etc., and there are few reports of studies on HAKAI protein function in mice at present, and no study on HAKAI in retinal tissues has yet been performed. Therefore, the specific action mechanism of HAKAI in the retina of mice is studied intensively, and the potential for treating and etiology discussing RP diseases is great.
The inventors of the present invention have found for the first time that knockout in retinal rod cells of target animalsHakaiGene or silencingHakaiThe gene is used for the gene expression,the target animal may be caused to exhibit RP disease-related characteristics. Thus, in retinal rod cellsHakaiThe animal with the knocked out or silenced gene can be used as an RP disease model, and can be used in the fields of RP disease research and the like, thereby providing a new model for research of the disease such as pathogenesis, mechanism and screening of related drugs.
In a preferred embodiment of the invention, the knockout isHakaiGene refers to knockoutHakaiAn exon sequence or a full-length sequence of a gene. Knock-outHakaiThe gene may be knocked outHakaiThe full-length sequence of the gene can also be knocked outHakaiPartial sequences of genes, e.g.partial exon sequences, whether sequences of the type knocked out (partial or full length), are those which are capable of being achieved in rod cellsHakaiKnock-out or silencing of genesHakaiThe expression of the gene makes animals show the corresponding characteristics of RP diseases, namely, the expression belongs to the protection scope of the invention.
A mouseHakaiGene (Casitas B-linear lymphoma-like 1, MGI: 2144842) is located on mouse chromosome 12 31,534,828-31,549,615bp, full length 14.787kb, its cDNA full length 4007bp, containing 6 exons.
In a preferred embodiment of the invention, the knockout isHakaiGene refers to knockoutHakaiAt least one exon sequence of exons 1 to 6 of the gene; one skilled in the art can select knockout as desiredHakaiAny one of exon 1, exon 2, exon 3, exon 4, exon 5 and exon 6 of the gene. Also can be knocked out selectivelyHakaiExons 1 and 2 of the gene; or knockout of exon 3 and exon 4; or knockout of exon 3, exon 4 and exon 5; or knockout of exon 5 and exon 6; or knockout of exons 1 to 6.
In an alternative embodiment, the knockout isHakaiGene refers to knockoutHakaiExon 3 to 5 sequences of the genes. The selection of exons 3 to 5 for knockout has good knockout efficiency.
In a preferred embodiment of the invention, the knockout is performed by at least one selected from the group consisting of CRISPR/Cas9 technology, CRISPR/Cas12a technology, CRISPR/Cas13a technology, artificial nuclease-mediated zinc finger nuclease technology, transcription activator-like effector nuclease technology and Cre-loxp gene knockout technology. However, in other embodiments, gene knockout is readily accomplished using gene editing techniques conventional in the art, provided that the knocked-out gene is specified.
In an alternative embodiment, the knockout is selected from the group consisting of CRISPR/Cas9 technology and Cre-loxp gene knockout technology.
In a preferred embodiment of the invention, the animal comprises a non-human target animalHakaiA gRNA vector or guide RNA complex of a gene, a Cas9 mRNA or Cas9 protein,HakaiThe homologous template sequence of the gene is transferred or chemically transfected into fertilized ovum of non-human target animal, embryo cells are taken and transplanted into uterus of pseudo-pregnant non-human target animal to obtainHakaiA first animal with conditional knockdown of the gene. The conversion includes, but is not limited to, electric conversion.
In an alternative embodiment, the first animal is mated and screenedHakaiConditional knockout of the gene into homozygous animals; and then will beHakaiHomozygous animals with conditional gene knockout and vectorsCreAnimal mating acquisition of genesHakaiA knockout animal;
in an alternative embodiment, the gRNA vector or guide RNA complex comprises a peptide for targetingHakaiA gRNA of the gene;Hakaihomologous template sequences of genes includeHakaiGenes, and inHakaiThe gene is provided with loxp sites at both ends.
In an alternative embodiment, the Cas9 protein is a Cas9 protein or a modified Cas9 protein. The modified Cas9 protein includes a functional domain in which an inhibitory activity such as EVE is added to the C-terminus of the Cas9 protein, and can specifically inhibit the expression of a target gene.
Further, in some embodiments of the invention, when the non-human animal is selected from a mouse, the above construction method comprises:
1) Will beObtained byHakai floxed Mating and breeding mice and wild mice, and screening offspringHakaiHeterozygote mice with conditional knockouts of genes;
2) The step 1) is carried outHakai floxed The heterozygote mice subjected to conditional gene knockout are bred by mating with each other to obtainHakaiA homozygous mouse is knocked out conditionally from the gene;
3) The step 2) is carried outHakaiThe homozygous mouse with conditional knockout gene is mated with the Rod-Cre gene transferred mouse, and the obtained offspringHakai floxed Rod-Cre andHakai floxed homozygote mating to obtain retina rod cellHakaiThe mouse animal with the gene knocked out can be used as a model of the retinal pigment degeneration disease.
In a preferred embodiment of the invention, the gene silencing technique is selected from RNAi technique or CRISPR/dCAs9 technique.
In a preferred embodiment of the invention, the method of gene silencing using CRISPR/dCas9 technology is as follows: will contain non-human target animalsHakaiThe gRNA vector or the guide RNA complex of the gene, dCAS9 mRNA or dCAS9 protein is transferred into fertilized eggs of non-human target animals through transformation or chemical transfection, embryo cells are taken and transferred into uterus of pseudopregnant non-human target animals, and the obtained product is obtainedHakaiAn animal with reduced gene expression; mRNA sequence of repression complex is connected to 3' end of dCAS9 mRNA or repression complex is connected to C end of dCAS9 protein;
in an alternative embodiment, the repressor complex is selected from the group consisting of Kruppel, H-NS (nuclear structured protein-like), stpA, LRP (leucine-reactive regulatory protein) or CRP (cAMP receptor protein) repressors. Such an embodiment can directly inhibitHakaiTranscription of genes, thereby achievingHakaiEffect of gene silencing.
In one example, the repressor is encoded by a prophage comprised by the host cell, such as an AcrIIA protein, e.g., acrIIA2 and/or AcrIIA4. It is also possible to code for the acr, the aca1 and the aca2 genes or their orthologues, homologs or paralogues or for the acrIIA2 and acrIIA4 genes or their orthologues, homologs or paralogues.
In one example, the CRISPR/Cas9 system is repressed by more than one such repressor (e.g., H-NS and LRP; or H-NS and CRP).
In an alternative embodiment, the non-human target animal is selected from the group consisting of non-human target mammals;
in an alternative embodiment, the non-human target mammal is selected from any one of a mouse, a rat, a rabbit, a cow, a dog, a pig, a horse, a sheep, a monkey, and a ape. Whatever animal is selected, so long as it hasHakaiAnimals of the genes, which are all the target (non-human mammal) animals in the above construction method of the present invention, are knocked out in their retinasHakaiThe gene shows the disease characteristics of retinal degeneration, is used as a retinal degeneration disease model in the field of retinal degeneration disease research, and belongs to the protection scope of the invention.
In a second aspect, the present invention also provides an application of the retinal pigment degeneration disease model constructed by the construction method described above in screening a drug for preventing or treating retinal degeneration disease, the drug having at least one of the following uses:
(1) If the candidate drug is administered, the vision of the model of retinal degenerative disease is improved compared to prior to administration of the candidate drug; indicating that the candidate drug may be used as a drug for preventing or treating a retinal degenerative disease;
(2) If the thickness of the outer and/or inner retinal layers is thicker or has a tendency to thicken after administration of the candidate drug than before administration of the candidate drug; indicating that the candidate drug may be used as a drug for preventing or treating a retinal degenerative disease;
(3) If the candidate drug is administered, the outer retinal segment increases compared to prior to administration of the candidate drug; indicating that the candidate drug may be used as a drug for preventing or treating a retinal degenerative disease;
(4) If the candidate drug is administered, the glioblast of the retina is reduced, the inflammatory response is reduced or eliminated, as compared to before the candidate drug is administered; it indicates that the candidate drug may be used as a drug for preventing or treating a retinal degenerative disease.
In a third aspect, the present invention also provides the use of a model of retinal degenerative disease constructed by the above-described method of constructing a model of retinal degenerative disease in the study of retinal degenerative disease, which is aimed at diagnosis or treatment of non-disease.
The animal model obtained by the construction method has typical RP disease characteristics and very wide application prospect, for example, the animal model is used for researching the pathogenesis of the RP disease, and provides a basis for deep understanding of the research on the RP disease. It can also be used for screening drugs for preventing or treating RP diseases, evaluating the efficacy or prognosis of drugs, etc.
In a fourth aspect, the present invention also provides a method for cultivating a model of a retinal degenerative disease, comprising the steps of: the retinal degenerative disease models obtained by the above-described construction methods are mated with each other.
After the RP disease model is obtained by the construction method for the first time, in order to obtain a larger number of RP disease models, a person skilled in the art can easily think that a breeding method for mutually mating RP disease models is used for obtaining a larger number of RP disease models, and the method for cultivating RP disease models also belongs to the protection scope of the invention.
The invention has the following beneficial effects:
the invention providesHakaiThe new application of the gene in the construction of the model of the retinal pigment degeneration disease. By inhibition ofHakaiExpression or genesHakaiThe genes are not expressed, so that the retina of the animal body can be damaged, and the outer nuclear layer and outer segment of the retina are abnormal. An animal model of a retinitis pigmentosa disease having a retinitis pigmentosa phenotype can be constructed. The invention provides a model foundation for the researchers in the field to deeply study the pathogenesis of the retinal pigment degeneration disease, and screen out the effective targeted therapeutic drugs in early clinic.
The invention also provides application of the animal model of the retinal pigment degeneration disease obtained by the model construction method in research of the retinal pigment degeneration disease, early molecular screening of drugs and screening of drugs for targeted treatment of retinal vascular diseases, and has very broad application prospects.
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: retinal rod cell knockoutHakaiConstruction route of gene mice (RKO);
fig. 2: primer 1 identifies the mouse result of the first generation by PCR; in the figure: primer pairs F1 and R1 are used for amplification, and the amplification product is MT:361bp, WT:308bp; wherein 23, 25, 26 are positive, WT: wild type; water: and (5) pure water control.HakaiGenotype identification results of the knockout mice; in the figureHakai floxed/floxed Representing homozygotes;Hakai +/+ refers to wild type;Hakai floxed /+ refers to heterozygotes; rod-Cre refers to Cre enzyme that specifically recognizes the loxp site in Rod cells;
fig. 3:Hakaidetecting gene knockout efficiency; a: real-time quantitative PCR experimental analysisHakaiKnockout efficiency in knockout mice retina; b, C: westernblot experimental analysisHakaiKnockout efficiency in knockout mice retina;
fig. 4: retinal rod cell knockoutHakaiGene mouse retina paraffin section H&E, dyeing results; a: specific knockdown of retinal rod cellsHakaiGene mouse retina paraffin section H&E, dyeing, namely thinning an outer nuclear layer; b: for a pair ofHakaiKnocking out the thickness statistics of the outer nuclear layer of the retina of the mouse; age of mice: 2 months;Hakai RKO representation ofHakaiA gene knockout mouse homozygote;Hakai Ctrl refers to wild type; ONL: outer nuclear layer (outer core layer); distance ON: distance to optic nerve (distance optic nerve)Distance); 2 mo represents 2 months of age;
fig. 5: specific knockdown of retinal rod cellsHakaiThe result of immunostaining of the gene mouse antibody shows thatHakaiShortening and degrading outer retinal joints of knockout mice, and age of the mice: for 2 months. DAPI (4', 6-diamidino-2-phenylindole); a nuclear dye, 4', 6-diamidino-2-phenylindole; rhodopsin, rhodopsin antibody; naK, sodium potassium ATPase antibodies; 2 mo represents 2 months of age;
fig. 6: specific knockdown of retinal rod cellsHakaiIHC staining results of the gene mice indicate thatHakaiThe retina of the knockout mouse is damaged, inflammatory reaction is initiated, and the age of the mouse is: 5 months; DAPI (4 ', 6-diamidino-2-phenylindole), a nuclear dye 4', 6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein antibody.
Detailed Description
Reference now will 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 to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols inMolecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.
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, knockoutHakaiThe route of the gene is shown in FIG. 1, and the operation steps are as follows:
1) We purchased C57BL/6J-Cbll1 from Sai industry Co em1Cflox Cya mice (abbreviated as Hakai) floxed ，https://www.cyagen.com/cn/zh-cn/sperm-bank-cn/S-CKO-00415）。
2) Hakai purchased in step 1) floxed The homozygous mouse with conditional knockout gene is mated with the Rod-Cre gene-transferred animal to obtain knockout in retina Rod cellsHakaiThe homozygous mouse of the gene can be used as a model of the retinal pigment degeneration disease. Rod-Cre transgenic mice (MGI: 4417915) were purchased from Jackson laboratories (Jackson Laboratory) USA. The Cre gene of the transgenic mouse is driven to express by a specific opsin promoter (Rod) in a retina Rod cell, cre protein can enter the nucleus of the Rod cell, and LoxP sites on a genome are identified, so that conditional knockout of the gene is realized.
Example 2
This example knocks out from retinal rod cellsHakaiHomozygous mice of the genes were identified.
The method comprises the following steps:
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. Mu.l of lysate (40 mM NaOH,0.2mM EDTA solution) was added to the centrifuge tube and heated at 100deg.C in the metal bath for 1h;
3) After taking out the centrifuge tube and cooling to room temperature, 100. Mu.l of a neutralization solution (40 mM Tris-HCl, pH 5.5) was added, 10000g was centrifuged for 2min, and the supernatant was used for genotyping of mice.
4) And (3) PCR amplification: the PCR reaction system was configured as follows
2×TaqMix10μL
Tail tissue lysate 2. Mu.L
Primer 1 (Hakai-loxP-Forward or Rod-Cre-Forward), 1. Mu.L (concentration: 10 mM);
primer 2 (Hakai-loxP-Reverse or Rod-Cre-Reverse), 1. Mu.L (concentration: 10 mM);
ddH 2 O 6μL。
the primer sequences were as follows:
Hakai-loxP-Forward sequence (SEQ ID NO. 1):
5’- AAGTATAGCGTGGTTTGCTGATTG -3’；
Hakai-loxP-Reverse sequence (SEQ ID NO. 2):
5’- GCTATAAAGCCACTTGTACCACAC -3’；
the Rod-Cre-Forward sequence (SEQ ID NO. 3):
5’-TCAGTGCCTGGAGTTGCGCTGTGG-3’；
Rod-Cre-Reverse sequence (SEQ ID NO. 4):
5’-CTTAAAGGCCAGGGCCTGCTTGGC-3’。
amplification procedure:
preheating: 94 ℃ for 3min; denaturation: 94 ℃, 30s, annealing: 62 ℃, 35s, extension: 72 ℃, 35s, circulation: 35 times; 72 ℃ for 5min; preserving at 4 ℃.
5) Gel electrophoresis
10ul of PCR product was taken in the wells and electrophoresed in 3% agarose gel at 120V constant pressure for 15min. Imaging was performed with a gel imaging system.
The results are shown in FIG. 2, aboveHakaiThe identification result of the gene knockout,Hakai +/+ representing a wild type control, the band size is 308bp;Hakai floxed/+ representing heterozygotes, having two bands of 308bp and 361bp respectively;Hakai floxed/floxed shows homozygote, has single band and has size of 361bp. The lower part is the identification result of the Rod-Cre gene, the Rod-Cre is 500bp, and the identification method can effectively identify the genotype of the mouse and select the gene with the following characteristics according to the result of FIG. 3Hakai floxed/floxed ； Rod-CreMice with the bands shown are models of retinal pigment degeneration disease.
Example 3
In this example, RT-QPCR and Westernblot experimental analysis were used, respectivelyHakaiGene knockout efficiency in gene knockout mice.
RT-QPCR experimental method:
1) Placing the mouse retina tissue in a 1.5ml centrifuge tube, adding 1ml of Trizol extract, and standing for 20 minutes at room temperature;
2) Adding 200ul of chloroform, fully and uniformly mixing, and standing at room temperature for 10 minutes;
3) Placing the sample in a 4-degree centrifuge, and centrifuging at 10000 revolutions for 15 minutes;
4) Carefully sucking the supernatant, adding equal volume of isopropanol, fully mixing, centrifuging at 10000 revolutions to precipitate RNA;
5) Washing the separated total RNA with 75% ethanol, centrifuging to precipitate again, air-drying, and adding DEPC water for dissolving;
6) cDNA was synthesized using total RNA as a template and then using cDNA synthesis kit (Invitrogen). According toHakaiPrimers were designed for cDNA sequences of (C). The primer sequences were as follows:
Hakai-cDNA-F(SEQ ID NO.5)：5’-TCAGCCCGTGGTATCTCAC-3'；
Hakai-cDNA-R(SEQ ID NO.6)：5’- GGTGGTGCGTAATGTTGCT-3'。
7) RT-QPCR was performed using the extracted cDNA as a template.
The results are shown in FIG. 4A. As can be seen, detection by RT-QPCR methodHakaiThe expression level was significantly reduced in the retina of RKO mice. In FIG. 4Hakai Ctrl Refers to the wild-type control, and,Hakai Rko refers to the retinal rod cells obtained in example 2HakaiHomozygous mice with knockouts, relative mRNA level, refer to relative changes in RNA expression levels, with wild type expression levels of 1.
Immunoblot (Western blot) experimental analysisHakaiGene knockout efficiency in gene knockout mice.
The method comprises the following steps: as in embodiment 1. The results are shown in FIGS. 3B and C, and it can be seen that the HAKAI content in the retina of RKO mice is significantly reduced, indicating that in RKO mice, HAKAI expression is silenced.
Example 4
This example uses retinal paraffin section H & E staining:
retinas of 2 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 2 months, the outer retinal nuclear layer of RKO mice had begun to thin, as compared to WT mice, indicating photoreceptor cell death (fig. 4).
Example 5
Immunohistochemical (IHC) Experimental analysis of this exampleHakaiThe retinas of the knockout mice were frozen and the length change of the outer retinal segments before and after knockout was compared by immunostaining the outer segment antibody Rhodopsin.
Immunostaining of frozen sections of retina: taking the construction of example 2 at 2 months of ageHakaiThe mice were knocked out, and after neck-breaking, the eyeballs were quickly taken and placed in 4% PFA, fixed on ice for 15min, then cut on cornea, and then fixed on ice. 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, the higher quality pieces were selected and placed in an oven at 37 ℃ for 30min, then the immunohistochemical pen was circled around the 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 (corresponding antibodies were selected depending on the target), 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.
As a result, as shown in FIG. 5, when the mice were 2 months old, the outer node antibody Rhodopsin was stained by frozen tissue sections of the retina, and it was found that the outer node of the retina of the RKO mice was significantly shortened and significantly degenerated as compared with the WT mice.
Example 6
Immune staining of glial cell marker GFAP in frozen sections of retina detects glial changes.
The results are shown in FIG. 6, where at 2 months of age the mice were dissected from retinal frozen tissueAfter plate staining of glial cell marker GFAP, it was found that,Hakaithe retina of the gene knockout mouse has obvious gliosis, and the inflammatory response is enhanced, which indicates retinal damage.
In summary, it can be seen that the embodiment of the invention takes mice as an example, and the retina rod cells are knocked outHakaiAfter the gene, the mice developed characteristics of retinal pigment-modified disease such as: visual impairment, shortening and degeneration of the extracellular node, loss of the visual cell, etc., are fully explained in the retinal rod cellsHakaiThe mouse after gene knockout can be used as a model of retinal pigment degeneration disease.
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 (10)
1. A method for constructing a model of a retinal pigment degeneration disease, comprising any one of the following steps:
(1) Knockout of the mouse retina rod cell genomeHakaiA gene;
(2) Gene silencing technology for mouse retina rod cell genomeHakaiThe expression level of the gene is reduced.
2. The method of claim 1, wherein the knockout is performedHakaiGene refers to knockoutHakaiAn exon sequence or a full-length sequence of a gene.
3. The method of construction according to claim 2, wherein the knockout is performedHakaiGene refers to knockoutHakaiAt least one of the exons 1 to 6 of the gene.
4. The method of claim 1, wherein the knockdown employs at least one selected from the group consisting of CRISPR/Cas9 technology, CRISPR/Cas12a technology, CRISPR/Cas13a technology, artificial nuclease-mediated zinc finger nuclease technology, transcription activator-like effector nuclease technology, and Cre-loxp gene knockdown technology.
5. The construction method according to claim 4, wherein the composition comprises a mouseHakaiA gRNA vector or guide RNA complex of a gene, a Cas9 mRNA or Cas9 protein,HakaiThe homologous template sequence of the gene is transferred or chemically transfected into fertilized ovum of mouse, embryo cells are transplanted into uterus of pseudopregnant mouse to obtainHakaiA first-established mouse with conditional gene knockout;
mating and screening first-established miceHakaiA homozygous mouse is knocked out conditionally from the gene; and then will beHakaiHomozygous mice with conditional gene knockout and carryingCreMouse mating of GeneHakaiA knockout mouse;
the gRNA vector or guide RNA complex comprises a peptide for targetingHakaiA gRNA of the gene; the saidHakaiHomologous template sequences of genes includeHakaiGenes, and inHakaiThe two ends of the gene are provided with loxp loci;
the Cas9 protein is a Cas9 protein or a modified Cas9 protein.
6. The method of claim 1, wherein the gene silencing technique is selected from RNAi technology or CRISPR/dCas9 technology.
7. The method of construction according to claim 6, wherein the gene silencing by CRISPR/dCas9 technique is performed as follows: will contain miceHakaiThe gRNA vector or the guide RNA complex of the gene, dCAS9 mRNA or dCAS9 protein is transferred into fertilized eggs of mice through transformation or chemical transfection, embryo cells are taken and transplanted into uterus of pseudopregnant mice, and the obtained product is obtainedHakaiMice with reduced gene expression; the 3' -end of dCAS9 mRNA is connected with mRNA sequence of repression complexOr the C-terminal of dCAS9 protein is connected with a repression complex;
the repressor complex is a Kruppel, H-NS, stpA, LRP or CRP repressor.
8. Use of the model of retinal pigment degeneration disease obtained by construction according to the construction method of any one of claims 1 to 7 for screening a drug for the prevention or treatment of retinal degeneration disease, characterized in that said drug has at least one of the following uses:
(1) If the candidate drug is administered, the vision of the model of retinal degenerative disease is improved compared to prior to administration of the candidate drug; indicating that the candidate drug may be used as a drug for preventing or treating a retinal degenerative disease;
(2) If the thickness of the outer and/or inner retinal layers is thicker or has a tendency to thicken after administration of the candidate drug than before administration of the candidate drug; indicating that the candidate drug may be used as a drug for preventing or treating a retinal degenerative disease;
(3) If the candidate drug is administered, the outer retinal segment increases compared to prior to administration of the candidate drug; indicating that the candidate drug may be used as a drug for preventing or treating a retinal degenerative disease;
(4) If the candidate drug is administered, the glioblast of the retina is reduced, the inflammatory response is reduced or eliminated, as compared to before the candidate drug is administered; it indicates that the candidate drug may be used as a drug for preventing or treating a retinal degenerative disease.
9. Use of a model of retinal degenerative disease constructed by the method of constructing a model of retinal degenerative disease according to any one of claims 1 to 7 in a study of retinal degenerative disease with the aim of diagnosis or treatment of non-disease.
10. A method for cultivating a model of a retinal degenerative disease, comprising the steps of: mating the model of retinal degenerative disease obtained by the construction method according to any one of claims 1 to 7 with each other.
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