Construction method of a prpf6 gene mutation-induced haploinsufficiency rp mouse model and application thereof

Abstract

The application discloses a method for constructing a RP mouse model caused by Prpf6 gene mutation and haploinsufficiency and application thereof, and belongs to the field of molecular biology and biomedical technology. The mouse model constructed by the method provides a convenient, reliable and economical means for studying the relationship between the PRPF6 gene mutation and the retinal pigment degeneration disease and the pathogenic mechanism thereof, and provides a stable hereditary effective research model in the research of the pathogenic mechanism, treatment method and drug screening of RP. The animal model is subjected to transcriptome and a series of type detection, wherein the transcriptome result shows that the retina of the Prpf6 gene knockout mouse has a certain degree of metabolic abnormality, and the results of the electro-physiological detection, fundus examination and H&E staining show that the cell layer of the mouse retina and the shape and function of the retina are damaged, which is consistent with the disease phenotype of RP.

Description

Technical Field

[0001] The present invention relates to a method for constructing a RP mouse model with haploinsufficiency caused by a Prpf6 gene mutation and an application thereof, and belongs to the technical field of molecular biology and biomedicine. Background Art

[0002] CRISPR / Cas9 technology is a rapidly developing gene editing tool with high precision and efficiency, widely used in fields such as genome editing, gene function research, and genetic disease treatment. The CRISPR / Cas9 system was first discovered in bacteria as a bacterial immune system, used to combat the invasion of foreign DNA. Its main components include sgRNA (single-guide RNA) and the Cas9 nuclease. The sgRNA guides Cas9 to a specific DNA target site and destroys the target DNA through Cas9's double-strand cleavage function. The mechanism of gene knockout relies on the cell's ability to repair double-strand breaks, typically through non-homologous end joining (NHEJ), resulting in base deletions or insertions, causing the target gene to lose function. The efficiency, simplicity, and specificity of CRISPR / Cas9 make it excellent for simultaneous editing of multiple gene loci. Despite challenges with off-target effects and repair efficiency, this technology has a wide range of applications, including gene function research, disease model development, and gene therapy.

[0003] Retinitis pigmentosa (RP) is a group of inherited retinal degenerative diseases characterized by progressive retinal photoreceptor apoptosis and retinal pigment epithelial degeneration, with significant clinical and genetic heterogeneity. In 2018, RP was included in my country's first list of rare diseases. The initial clinical manifestations of RP are night blindness and constriction, typically beginning during adolescence. Subsequently, with the irreversible degeneration of cones and rods, complete blindness eventually develops. Currently available clinical medications and treatments are ineffective in delaying or treating RP. Most RP-causing genes are specifically expressed in the retina and are involved in various functions of the photoreceptor system, including phototransduction signaling, retinal transcription factor pathways, retinal metabolism, cellular structure, and ciliary structure and function. Among these, six small nuclear ribonucleoprotein particles (snRNPs) involved in splicing (PRPF3, PRPF4, PRPF6, PRPF8, PRPF31, and SNRNP200) can also cause RP. In addition, we previously performed exome sequencing on over 200 patients with retinitis pigmentosa and discovered several novel snRNP gene mutations, including a novel splice site mutation in the PRPF6 gene (c.2673+1G>A). Currently, most splicing factor mutations that cause RP are inherited in an autosomal dominant manner, and the cause of the disease can be attributed to haplodeficiency caused by loss of function (LOF) mutations. However, existing studies on RP caused by PRPF6 mutations are limited, and further research is needed to understand the molecular mechanisms of RP caused by PRPF6 mutations.

[0004] The PRPF6 gene (Pre-mRNA Processing Factor 6) encodes a protein associated with the spliceosome and is involved in the splicing process of pre-mRNA in eukaryotic cells. Studies in recent years have shown that mutations in PRPF6 are closely related to RP. The protein encoded by the PRPF6 gene is involved in the splicing of pre-mRNA and is a key component of the spliceosome U4 / U6-U5 triad. Mutations in PRPF6 disrupt the normal function of the spliceosome, leading to abnormal mRNA splicing in retinal photoreceptor cells, triggering protein production errors, and then leading to the degeneration of these cells. Current research is exploring potential treatments such as gene therapy and RNA splicing correction, but more research and clinical verification are still needed. Research on PRPF6 and RP not only reveals the pathogenic mechanism of spliceosome dysfunction, but also provides an important direction for the future treatment of RP.

[0005] Based on the above characteristics of PRPF6 and RP, although existing technologies have reported the correlation between PRPF6 gene knockout and RP, due to the genetic complexity of RP, although the above mouse model can simulate related symptoms similar to RP to a certain extent, the research results are relatively limited, and it is difficult to approach the ideal disease model of RP caused by PRPF6 mutations, and thus it is difficult to use it for research on gene therapy of RP caused by PRPF6 mutations. Summary of the Invention

[0006] The present invention provides a method for preparing a mammalian model of retinitis pigmentosa caused by Prpf6 gene mutation. The method comprises: removing exons 4 to 11 of the Prpf6 gene from a mouse homologous chromosome without affecting upstream and downstream genes, forming a deletion of approximately 17 kb in length, with a coding sequence of 1165 bp, accounting for 41.27% of the CDS, and the knocked-out coding sequence is not a multiple of 3, resulting in a frameshift mutation, thereby destroying the function of the gene, thereby achieving the knockout of Prpf6.

[0007] The present invention provides a method for preparing a non-human mammal model of retinitis pigmentosa (RP), which comprises: removing exons 4 to 11 of the Prpf6 gene in the homologous chromosome of the animal to achieve gene knockout.

[0008] According to the structure of the Prpf6 gene, exon4-11 of the Prpf6 gene was selected as the knockout region. It should be noted that although we selected this region as the knockout region, the genome upstream and downstream of the knockout region in mice are all introns, and the absence of introns has no effect on the construction of the model of the present invention. Therefore, the knockout region of the present invention can be the region between exon3-12 of the Prpf6 gene, which does not include exon3 exon and exon12 exon, and must include exon4-exon11 region.

[0009] In one embodiment of the present invention, the Prpf6 gene can refer to NM_133701.2 (NCBI RefSeq), where NM_133701.2 is the complete mature mRNA that has been sheared and processed; the sequence with Ensembl ID ENSMUSG00000002455 can also be referred to; and the transcript of Prpf6201: ENSMUST00000002529 can also be referred to.

[0010] In one embodiment of the present invention, exons 4 to 11 of the Prpf6 gene are excised using the CRISPR / Cas9 system.

[0011] In one embodiment of the present invention, the CRISPR / Cas9 system includes sgRNA and Cas9 enzyme, and the sgRNA sequences are shown in SEQ ID NOs. 1 to 4, respectively.

[0012] In one embodiment of the present invention, the object of gene knockout is the Prpf6 gene of the fertilized egg.

[0013] In one embodiment of the present invention, the method for introducing the CRISPR / Cas9 system into the fertilized egg is microinjection.

[0014] In one embodiment of the present invention, the method further comprises transplanting the treated fertilized eggs into pseudo-pregnant female animals to produce F0 generations, and performing genotyping to obtain retinitis pigmentosa (RP) disease animals.

[0015] In one embodiment of the present invention, the non-human mammal is a rodent.

[0016] In one embodiment of the invention, the non-human mammal is a mouse.

[0017] In one embodiment of the present invention, the method comprises: injecting the RNP injection complex into a mouse fertilized egg, transplanting the fertilized egg into a surrogate mouse, and producing a RP disease mouse.

[0018] In one embodiment of the present invention, the RNP injection complex is prepared by adding sgRNA to water, mixing and incubating, and then adding Cas9 protein, mixing and incubating to obtain the RNP injection complex.

[0019] In one embodiment of the present invention, the sgRNA and Cas9 protein are added in a volume ratio of 7:1.

[0020] In one embodiment of the present invention, the sgRNA is obtained by mixing sgRNAs with sequences of SEQ ID NOs. 1 to 4.

[0021] In one embodiment of the present invention, the RNP injection complex is prepared by adding 1.4 μL of 100 pmol / μL sgRNA (obtained by mixing sgRNAs with sequences of SEQ ID NOs. 1 to 4 at a ratio of 1:1:1:1 (v / v / v / v)) to 5.2 μL of RNase-free water, mixing and incubating for 5 minutes, and then adding 0.2 μL of Cas9 protein and mixing and incubating for 10 minutes to obtain an RNP injection complex.

[0022] The present invention also provides a mouse model of RP disease caused by haploinsufficiency due to Prpf6 gene mutation. In the mouse model, exons 4 to 11 of the Prpf6 gene in the mouse homologous chromosome are removed, resulting in a frameshift mutation, destroying the gene function, and achieving knockout of Prpf6.

[0023] In one embodiment of the present invention, the mouse model utilizes non-homologous end joining to achieve knockout of exons 4 to 11 of the Prpf6 gene through the CRISPR / Cas9 system.

[0024] In one embodiment of the present invention, the CRISPR / Cas9 system includes sgRNA and Cas9 enzyme, and the sgRNA sequences are shown in SEQ ID NOs. 1 to 4, respectively.

[0025] In one embodiment of the present invention, the RP disease is caused by haploinsufficiency due to loss-of-function mutation.

[0026] The present invention also provides a method for preparing a mouse model of RP disease with haploinsufficiency caused by Prpf6 gene mutation, the method comprising: using the CRISPR / Cas9 system to excise exons 4 to 11 of the mouse Prpf6 gene.

[0027] In one embodiment of the present invention, the method specifically comprises the following steps:

[0028] The sgRNA sequences shown in SEQ ID NOs. 1 to 4 and the Cas9 protein are mixed to obtain an RNP injection complex, which is then injected into a mouse fertilized egg. The fertilized egg is then transplanted into a surrogate mouse, and mice are born and genotyped to obtain F0 generation mice with successful gene knockout, i.e., RP disease mice;

[0029] The F0 generation mice with successful gene knockout were crossed with wild-type mice to obtain F1 generation heterozygous gene knockout mice, i.e. RP disease mice;

[0030] The obtained F1 generation heterozygous male and female mice were mated with each other to obtain F2 generation heterozygous gene knockout mice, i.e. RP disease mice;

[0031] The obtained F2 generation heterozygous male and female mice were mated with each other to obtain Prpf6 gene knockout heterozygous mice, namely the RP disease mouse strain.

[0032] In one embodiment of the present invention, the RNP injection complex is prepared by adding sgRNA to water, mixing and incubating, and then adding Cas9 protein, mixing and incubating to obtain the RNP injection complex.

[0033] In an embodiment of the present application, the sgRNA and Cas9 protein are added in a ratio of 7:1 by volume.

[0034] In an embodiment of the present application, the sgRNA is obtained by mixing sgRNAs with sequences of SEQ ID NO. 1-4 in a ratio of 1:1:1:1 (v / v / v / v).

[0035] In an embodiment of the present application, the RNP injection complex is obtained by adding 1.4 μL of 100 pmol / μL sgRNA (obtained by mixing sgRNAs with sequences of SEQ ID NO. 1-4 in a ratio of 1:1:1:1 (v / v / v / v)) in 5.2 μL of RNase-free water, mixing and incubating for 5 min, then adding 0.2 μL of Cas9 protein, mixing and incubating for 10 min.

[0036] The present application also provides a method for preparing a Prpf6 gene knockout mouse model, which comprises: using a CRISPR / Cas9 system to remove the 4th to 11th exon of the mouse Prpf6 gene.

[0037] In an embodiment of the present application, the CRISPR / Cas9 system comprises sgRNA and Cas9 enzyme, and the sgRNA sequences are shown in SEQ ID NO. 1-4.

[0038] In an embodiment of the present application, the method specifically comprises the following steps:

[0039] The sgRNA sequences shown in SEQ ID NO. 1-4 and Cas9 protein are mixed to obtain an RNP injection complex, the RNP injection complex is injected into a mouse zygote, the zygote is transplanted into a surrogate mouse, a mouse is produced and genotyped to obtain a F0 generation mouse with successful gene knockout;

[0040] The F0 generation mouse with successful gene knockout is crossed with a wild type mouse to obtain a F1 generation hybrid gene knockout mouse;

[0041] The F1 generation hybrid male and female mice obtained are mated to obtain a F2 generation hybrid gene knockout mouse;

[0042] The F2 generation hybrid male and female mice obtained are mated to obtain a Prpf6 gene knockout hybrid mouse strain.

[0043] In an embodiment of the present application, the RNP injection complex is obtained by adding sgRNA in water, mixing and incubating, then adding Cas9 protein, mixing and incubating.

[0044] In one embodiment of the present invention, the sgRNA and Cas9 protein are added in a volume ratio of 7:1.

[0045] In one embodiment of the present invention, the sgRNA is obtained by mixing sgRNAs with sequences of SEQ ID NOs. 1 to 4.

[0046] In one embodiment of the present invention, the RNP injection complex is prepared by adding 1.4 μL of 100 pmol / μL sgRNA (obtained by mixing sgRNAs with sequences of SEQ ID NOs. 1 to 4 at a ratio of 1:1:1:1 (v / v / v / v)) to 5.2 μL of RNase-free water, mixing and incubating for 5 minutes, and then adding 0.2 μL of Cas9 protein and mixing and incubating for 10 minutes to obtain an RNP injection complex.

[0047] The present invention also provides a Prpf6 gene knockout mouse model, which is prepared by the above method.

[0048] The present invention also provides the use of the above-mentioned Prpf6 gene knockout mouse model in studying the pathogenic mechanism of RP.

[0049] The present invention also provides the use of the Prpf6 gene knockout mouse model in studying RP gene therapy methods.

[0050] The present invention also provides a CRISPR / Cas9 system for excising exons 4 to 11 of the mouse Prpf6 gene, wherein the CRISPR / Cas9 system comprises sgRNA and Cas9 enzyme, and the sgRNA sequences are shown in SEQ ID NOs. 1 to 4, respectively.

[0051] The present invention also provides the use of the above-mentioned Prpf6 gene mutation-induced haploinsufficient RP disease mouse model or the mouse model prepared by the above-mentioned method in screening drugs for preventing or treating retinitis pigmentosa.

[0052] The present invention also provides the use of the above-mentioned Prpf6 gene mutation-induced haploinsufficient RP disease mouse model or the mouse model prepared by the above-mentioned method in RP disease research, wherein the research is for the purpose of non-disease treatment.

[0053] The present invention also provides use of the above mouse model, or the mouse model obtained by the above method, in preparing drugs for identifying and / or testing retinitis pigmentosa.

[0054] In one embodiment of the present invention, the drug is used to prevent and / or treat retinitis pigmentosa, and / or treat complications associated with retinitis pigmentosa.

[0055] The present invention also provides a method for cultivating a mouse model of retinitis pigmentosa, which comprises the following steps: mating the above-mentioned RP disease mouse model with haploinsufficiency caused by the Prpf6 gene mutation or the mouse model prepared by the above method to obtain heterozygous mice.

[0056] Beneficial effects:

[0057] Compared with the prior art, the present invention has the following significant advantages:

[0058] (1) The present invention provides a method for constructing a mouse model of retinitis pigmentosa caused by Prpf6 mutation. The mouse model constructed using this method can be stably propagated, providing a convenient, reliable and economical means for studying the relationship between PRPF6 gene mutation and retinitis pigmentosa and its pathogenic mechanism.

[0059] (2) In the case that human patient research materials are not easily available and are subject to medical ethical constraints, the mouse model provided by the present invention will become an important tool in the study of retinitis pigmentosa associated with PRPF6 mutation.

[0060] (3) The mouse model provided by the present invention provides an effective research model with stable inheritance in the study of pathogenic mechanisms, treatment methods, drug screening, etc., especially providing a mouse model of retinitis pigmentosa caused by Prpf6 mutation in gene editing-based treatment methods.

[0061] (4) The construction method of the present invention provides a basis for studying the related factors and diagnosis and treatment of PRPF6 mutations in humans.

[0062] (5) The method of the present invention can affect the overall expression of the Prpf6 gene, thereby achieving the effect of haploinsufficiency of the splicing factor, which can be relatively closer to the disease model of RP caused by Prpf6 mutation.

[0063] (6) Compared with other retinitis pigmentosa disease model mice, the mouse model provided by the present invention has a slow phenotype and a robust development process, allowing researchers to observe the progression and changes of the disease over a long period of time and gain a deeper understanding of the nature of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Schematic diagram of the construction strategy for the Prpf6 gene mutation mouse model using CRISPR / Cas9 gene targeting technology.

[0065] Figure 2Schematic diagram of the primer design strategy.

[0066] Figure 3 Agarose gel electrophoresis diagram of gene identification of F0 generation mice.

[0067] Figure 4 This is the sequencing result diagram for gene identification of F0 generation mice.

[0068] Figure 5 Schematic diagram of the primer design strategy.

[0069] Figure 6 Schematic diagram of the mouse breeding route.

[0070] Figure 7 Agarose gel electrophoresis diagram for gene identification of F1 generation mice.

[0071] Figure 8 This is the GO enrichment result diagram of the transcriptome sequencing of the Prpf6 gene knockout mouse model.

[0072] Figure 9 This is the KEGG enrichment result diagram of the transcriptome sequencing of the Prpf6 gene knockout mouse model.

[0073] Figure 10 For wild type and Prpf6 + / - Quantitative statistical graphs of mouse retinal ERG detection; Figure A represents the amplitude of the a wave, Figure B represents the time when the a wave appears, Figure C represents the amplitude of the b wave, and Figure D represents the time when the b wave appears.

[0074] Figure 11 For wild type and Prpf6 + / - Fundus photography of mice; the left picture is a wild-type mouse fundus photography; the right picture is Prpf6 + / - Fundus photography of mice.

[0075] Figure 12 Prpf6 + / - H&E staining results of mouse eye cup. DETAILED DESCRIPTION

[0076] The experimental animal strain involved in the following embodiments is: C57BL / 6J, and the surrogate mother mouse strain is C57BL / 6J, which was purchased from Saiye (Suzhou) Biotechnology Co., Ltd.

[0077] Example 1: Construction of Prpf6 gene knockout mouse model

[0078] In this example, CRISPR / Cas9 technology was used to achieve exon 4 to 11 excision of the mouse Prpf6 gene by non-homologous end joining, resulting in a frameshift mutation, to obtain a Prpf6 gene knockout mouse model. The specific strategy is shown in Figure 1;like Figure 1 As shown, the non-homologous end joining region for achieving gene knockout is upstream of exon 4 and downstream of exon 11 of the Prpf6 gene.

[0079] In this example, C57BL / 6J mice were used to construct a Prpf6 gene knockout mouse model; the C57BL / 6J mice used were purchased from Saiye (Suzhou) Biotechnology Co., Ltd. and were 8 weeks old.

[0080] In this example, sgRNAs targeting regions upstream of exon 4 and downstream of exon 11 of the mouse Prpf6 gene were synthesized. These sgRNAs were then co-injected with Cas9 protein into fertilized mouse eggs to produce F0 offspring with the targeted knockout. The genotypes of the F0 animals were identified by PCR and sequence analysis, and the animals were then co-bred with wild-type mice to detect germline transmission and generate heterozygous animals, establishing a mouse model of retinitis pigmentosa.

[0081] The specific construction process is as follows:

[0082] 1. Gene knockout target site design

[0083] (1) Genetic information:

[0084] The Prpf6 gene (https: / / www.ncbi.nlm.nih.gov / gene / 68879, NCBI reference sequence: NM_133701.2; Ensembl: ENSMUSG00000002455) is located on chromosome 2 of mice. The gene has 21 exons, with the first exon containing an ATG start codon and the 21st exon containing a TGA stop codon. The transcript is Prpf6201: ENSMUST00000002529 transcript, and the knockout region is exon4-11.

[0085] The sequence of the knockout region is as follows:

[0086]

[0087] The uppercase part in the above gene sequence represents exons 4 to 11 in the mouse Prpf6 gene, and the lowercase part represents the intron region.

[0088] (2) CRISPR / Cas9 gene knockout target site design

[0089] Using the online sgRNA design platform and sgRNA design principles provided by http: / / crispor.tefor.net / , we evaluated the target sites with high scores near the upstream exon 4 and downstream exon 11 of the mouse Prpf6 gene and designed sgRNAs. The designed sgRNA sequences are shown in SEQ ID NOs. 1 to 4:

[0090] SEQ ID NO.1:

[0091] sgRNA-A1:GACATCTATGGGCACTAGGA-AGG

[0092] SEQ ID NO.2:

[0093] sgRNA-A2:CGCGAGACCTTATGTAGAAG-AGG

[0094] SEQ ID NO.3:

[0095] sgRNA-B1:TCCACTGCTCCAGTTCTAAG-GGG

[0096] SEQ ID NO.4:

[0097] sgRNA-B2: CACTCACAATGAGCCCGTGT-GGG

[0098] The above sgRNA matches the antisense strand of the gene. The dash represents the PAM sequence. The full name of the PAM sequence is Protospacer adjacent motif, which means original spacer adjacent motif in Chinese.

[0099] 2. Preparation of ribonucleoprotein (RNP) complexes

[0100] First, sgRNA solutions of sgRNA sequences shown in SEQ ID NOs. 1 to 4 were synthesized by GenScript Biotech Co., Ltd.

[0101] Then, 1.4 μL of 100 pmol / μL mixed sgRNA (obtained by mixing the sgRNA solutions of SEQ ID NOs. 1 to 4 at a ratio of 1:1:1:1 (v / v / v / v)) was added to 5.2 μL of RNase-free water, mixed and incubated for 5 minutes, and then 0.2 μL of Cas9 protein (NEB, Catalog No.: M0646M) was added and mixed and incubated for 10 minutes to obtain an RNP injection complex.

[0102] 3. Microinjection of fertilized eggs and embryo transfer

[0103] (1) Four-week-old C57BL / 6J female mice were selected and injected with 0.1 mL of pregnant mare serum (PMSG). 48 hours later, 0.1 mL of human chorionic gonadotropin (HCG) was injected. After HCG injection, the female mice were mated with adult fertile male mice to fertilize them. The presence of mating was determined by examining the vaginal plug.

[0104] The next day, the female mice were euthanized and the fertilized eggs were collected from the oviduct.

[0105] (2) Use a microinjector to inject the RNP injection complex prepared in step 2 above into the nucleus of a mouse fertilized egg. Transfer the injected fertilized egg to M16 culture medium (purchased from Sigma, product number: M7292) and place it in a 37°C constant temperature 5% CO2 incubator. Culture until the 2-cell stage and wait for transplantation the next day.

[0106] (3) selecting fertile female mice of appropriate age to mate with male mice that have been sterilized after vasectomy, stimulating the female mice to undergo a series of pregnancy changes to obtain pseudo-pregnant female mice, which serve as surrogate mice after the fertilized eggs are genetically modified; transplanting the fertilized eggs injected with the RNP injection complex obtained in step (2) into the oviduct of the surrogate female mice on the day of thrombosis;

[0107] After the oviduct transplantation was successful, the mother mouse was waited for to give birth to F0 mice and PCR identification and genotyping were performed to obtain four heterozygous mice with successful knockout. The F0 mice were numbered as 3, 4, 12, and 16 respectively.

[0108] 4. Genotype identification of F0 generation mice

[0109] The identification of F0 generation mice is as follows:

[0110] (1) Genomic DNA extraction

[0111] Genomic DNA from F0 mice was extracted using an alkaline lysis method. First, tail tip tissue from a 2-week-old F0 mouse was collected and placed in a 1.5 mL EP tube. 100 μL of Solution A was added, followed by incubation at 100°C in a dry-well incubator for 30 minutes. Finally, 100 μL of Solution B was added, the tube was thoroughly vortexed and centrifuged. The resulting genomic DNA was then stored in a refrigerator at 4°C until ready for use. Solution A consisted of 25 mmol / L NaOH and 2 mmol / L EDTA; Solution B consisted of 40 mmol / L Tris·HCl, pH 8.0.

[0112] (2) PCR amplification

[0113] A pair of PCR primers (F1 / R1) were designed for the upstream and downstream sequences of the knockout fragment (Table 1). Figure 2 25 μL of PCR mixture was prepared according to the standard PCR system, and the number of PCR cycles was set to 32 (Table 2); wherein, the Premix Taq Polymerase used was purchased from Vazyme (P222).

[0114] PCR genotyping uses two sets of controls:

[0115] Negative control (WT): wild-type mouse genomic DNA was used instead of the F0 generation mouse genomic DNA;

[0116] Blank control (Water): ddH2O was used instead of the genomic DNA of the F0 generation mice.

[0117] A 668 bp fragment was obtained by PCR amplification of mouse DNA using F1 / R1, indicating that exons 4 to 11 of the Prpf6 gene were successfully knocked out;

[0118] The gene fragment obtained by PCR amplification of wild-type mouse DNA using F1 / R1 was 17942bp in size.

[0119] Table 1 Primer sequences

[0120]

[0121] Table 2 PCR reaction system

[0122]

[0123]

[0124] (3) Identification results

[0125] Mice No. 3, 4, 12, and 16 were positive after the target gene was deleted. The results of agarose gel electrophoresis were as follows: Figure 3 shown.

[0126] The DNA product obtained by PCR amplification in step (2) was sent for first-generation sequencing to confirm the knockout status.

[0127] Sequencing primers:

[0128] F1 (SEQ ID NO.5): 5'-TACAGGTGTATGTTACTTTGCCTGAC-3'

[0129] R1 (SEQ ID NO.6): 5'-CTGGTTTGCTTTAGCAAGGGTA-3'

[0130] Sequencing results:

[0131] The sequencing results of mice No. 3, 4, 12, and 16 were as follows: the length of the Prpf6 gene sequence was 17274 bp (the length after deleting 17942 bp), and the Prpf6 gene exons 4 to 11 were knocked out, that is, the Prpf6 gene was successfully knocked out. The results are as follows Figure 4 shown.

[0132] 5. Obtaining and identifying F1, F2, and Prpf6 gene knockout heterozygous mouse lines

[0133] Examples of breeding routes include Figure 6 As shown. By self-crossing (+ / -) heterozygous mice, (- / -) homozygous mice, (+ / -) heterozygous mice, and (WT) wild-type mice can be generated. The following primer sets are used to determine the genotype. PCR identification strategy is as follows Figure 5 shown.

[0134] Primer set 1:

[0135] F1 (SEQ ID NO.5): 5'-TACAGGTGTATGTTACTTTGCCTGAC-3'

[0136] R1 (SEQ ID NO.6): 5'-CTGGTTTGCTTTAGCAAGGGTA-3'

[0137] When the target band (668 bp fragment) was amplified by PCR on mouse DNA using primer set 1, it was determined that the target gene was successfully deleted.

[0138] Primer set 2:

[0139] F2 (SEQ ID NO.7): 5'-GCTTTCACTCAGGGTTCTGTTCTAA-3'

[0140] R1 (SEQ ID NO. 6): 5'-CTGGTTTGCTTTAGCAAGGGTA-3'

[0141] PCR amplification of the target band (865bp fragment) from mouse DNA using primer set 2 indicates that the target gene has not been successfully deleted.

[0142] Using F1, R1, F2 primers to determine the genotype of (- / -) homozygotes, (+ / -) heterozygotes, and (+ / +) wild-type mice, the amplification results are as follows:

[0143] The target band of (- / -) homozygote genotype is 668bp;

[0144] The target band of (+ / -) heterozygote genotype is 668bp and 865bp;

[0145] The target band of (WT) wild-type genotype is 865bp.

[0146] Due to the importance of Prpf6 gene for mouse growth and development, the present application found that it was not possible to breed Prpf6 gene knockout homozygotes in subsequent breeding, and it was speculated that Prpf6 gene knockout homozygote mice may not survive. Heterozygote genotype mice are Prpf6 gene knockout mice (Prpf6 + / - mice) obtained by the present application.

[0147] (1) The obtaining and identification process of F1 generation mice is as follows:

[0148] The above-mentioned F0 generation male mice with successful knockout of the target gene were crossed with wild-type female mice, and after 1 week, the pregnant female mice were raised in single cages, and F1 generation mice were obtained after delivery. After 3 weeks of birth, the tail of F1 generation mice was cut to extract tissue DNA, which was detected by PCR reaction and agarose gel electrophoresis to screen out Prpf6 gene knockout mice. The specific operation is the same as the identification method of F0 generation gene knockout mice described above, the primers are shown in Table 3, and the PCR reaction conditions are shown in Table 4.

[0149] The PCR identification results are shown in Figure 7 , and Prpf6 gene knockout heterozygote mice (Prpf6 + / - mice) are obtained.

[0150] Table 3 primer sequences

[0151]

[0152] Table 4 PCR reaction system

[0153]

[0154] (2) Obtaining F2 generation Prpf6 gene knockout heterozygous mice

[0155] The F1 generation heterozygous mutant female mice obtained in step (1) above were caged with heterozygous mutant male mice. One week later, the pregnant female mice were housed individually and gave birth to obtain F2 generation hybrid mice. Three weeks after the birth of the F2 generation hybrid mice, the tails were cut and tissue DNA was extracted. Prpf6 gene knockout mice were screened by PCR reaction and agarose gel electrophoresis. The specific operation was the same as the F1 generation gene knockout mouse genotype identification method in step (1) above. The primers used were shown in Table 3, and the PCR reaction conditions were shown in Table 4.

[0156] (3) Obtaining Prpf6 gene knockout heterozygous mouse strain

[0157] From the F2 generation of mutants, heterozygous female and male mice were selected. After being housed together for one week, the pregnant females were housed individually and gave birth to F3 mice. Three weeks after birth, one mouse from each litter was selected, tail snipped, and DNA extracted from tissue. Genotypes were identified by PCR amplification and agarose gel electrophoresis to identify Prpf6 knockout mice. The specific procedures were the same as those for the knockout mouse genotyping method described above. The primers used were listed in Table 3, and the PCR reaction conditions are shown in Table 4.

[0158] The obtained F3 generation heterozygous mice were housed together, bred and expanded, and the Prpf6 gene knockout heterozygous mouse strain (Prpf6 + / - mice).

[0159] Example 2: Molecular phenotype detection of Prpf6 gene knockout mouse model - transcriptome sequencing

[0160] Three 6-month-old wild-type mice and three Prpf6 gene knockout heterozygous mice obtained in Example 1 were selected. After removing all blood from the mice by blood perfusion, the eyeballs of the mice were removed and placed in physiological saline. Then, the retinas were detached using ophthalmic scissors and ophthalmic forceps under a stereomicroscope.

[0161] Retinal tissue was digested at room temperature using 1 mL of Trizol reagent (purchased from Kangwei Century). After complete tissue digestion, 200 μL of chloroform was added and mixed thoroughly. The tube was then centrifuged at 1200 rpm for 15 minutes at 4°C. 500 μL of the supernatant was transferred to a new 1.5 mL RNase-free EP tube and 500 μL of isopropanol was added to precipitate RNA. After mixing thoroughly, the tube was centrifuged at 1200 rpm for 5 minutes at 4°C. The supernatant was carefully removed and the pellet was washed with 1 mL of 75% ethanol. The pellet was also centrifuged at 1200 rpm for 5 minutes at 4°C. The supernatant was carefully removed and the tube was air-dried at room temperature for 10 minutes. 30 μL of RNase-free water was added to dissolve the RNA.

[0162] The obtained RNA was sent to Beijing Novogene Co., Ltd. for transcriptome sequencing, and the sequencing results were subjected to transcriptomic analysis. The differentially expressed genes in the retina of wild-type mice and heterozygous mice were subjected to functional enrichment analysis.

[0163] The results are as follows Figure 8 As shown in the results, GO (Gene Ontology Enrichment Analysis) analysis results showed that these differentially expressed genes were mainly involved in the decomposition and metabolism of hydrogen peroxide, the metabolism of reactive oxygen species, cell connection, the maintenance of extracellular matrix tissue structure and function, oxygen carrier activity, peroxidase activity, and the regulation of oxidoreductase activity. KEGG (Kyoto Encyclopedia of Genes and Genomes) results showed that these genes were involved in the TGF-β signaling pathway related to cell growth, differentiation, and migration, the leukocyte transendothelial migration pathway related to inflammation ( Figure 9 The functions and pathways mentioned above are all closely related to the retina, indicating that the retina of Prpf6 knockout mice has undergone a certain degree of metabolic abnormalities, which to some extent verifies the Prpf6 knockout mouse model.

[0164] Example 3: Retinal function detection in Prpf6 gene knockout mouse model

[0165] (1) Electrophysiological examination of mouse ophthalmology (ERG)

[0166] Five-month-old wild-type mice and Prpf6 knockout mice (Prpf6 + / - Five mice were gently removed from their cages and sent to Beijing Xinlian Optoelectronics for ERG examination. Mice were anesthetized with Avertin (tribromoethanol) (Meilun Biotechnology Co., Ltd.) at a standard anesthetic dosage of 30 μL / g.

[0167] The parameters examined were as follows: rod response (Rod): 0.015 log cd s / m2; cone response (Cone): 3.0 log cd s / m2; maximum mixed response (dmax): 3.0 log cd s / m2.

[0168] The inspection results found that:

[0169] Prpf6 + / - The peak amplitude of the a-wave of rod cells in the mouse retina decreased significantly, while the peak amplitude of the a-wave of cone cells did not change significantly; compared with wild-type mice, Prpf6 + / - There was no significant change in the a-wave duration of rods and cones in the mouse retina.

[0170] The peak amplitude of the b wave showed that Prpf6 + / - The b-wave peaks of rods and cones in the mouse retina decreased significantly; the b-wave duration of rods and cones in the mouse retina did not change significantly.

[0171] The results showed that the peak amplitudes of a-wave and b-wave decreased significantly. Figure 10 As shown, this suggests that Prpf6 + / - The mouse retinal cell layers were severely damaged.

[0172] (2) Fundus examination of mice

[0173] After the ERG examination was completed, the mice were awakened from anesthesia and recovered for 3 days. The fundus of the mice was then photographed. Anesthesia and pupil dilation were performed in the same way. Most Prpf6 + / - There is no obvious pigmentation in the fundus of mice, but there are white spots, which may be related to the change of pigment ( Figure 11 ).

[0174] (3) H&E staining

[0175] After fundus photography, mice in each group were anesthetized and supplemented with 4-week-old Prpf6 + / - Mice were used as a control group for disease progression and perfused through the left ventricle with 30 mL of normal saline. The eyeballs were then removed and fixed in a modified Davidson's fixative (A) purchased from Regen Biotech. After 2 hours, the lens was removed from the cornea to facilitate later sectioning. After the lens was removed, it was placed back in the modified Davidson's fixative (A) and fixed at 4°C for 48 hours. After fixation, the eye cup was removed and placed in an embedding box, rinsed with running water for 1 hour, and then immersed in 75%, 85% and 95% ethanol for 30 minutes each, and finally immersed in n-butanol solution for 1 hour. After dehydration, it was waxed and embedded. After embedding, paraffin sections were made to obtain 4 μm thick sections. The fixed sections were naturally air-dried.

[0176] Subsequent H&E staining uses the non-toxic and environmentally friendly hematoxylin-eosin stain (Cat. No. D006-1) from Nanjing Jiancheng Technology Co., Ltd. The specific steps are as follows:

[0177] First, dewax the paraffin sections using a non-toxic and environmentally friendly dewaxing agent for 15 minutes each time, for a total of two times; then immerse the sample slides in 95%, 70%, and 30% ethanol for 2 minutes each, and in warm water for 2 minutes; remove them from the warm water and shake off excess water to allow the distillation to evenly wet the entire tissue, place them in the nuclear stain hematoxylin for about 5 minutes, and wash with water for 5 seconds; then place them in the pulp stain eosin for about 15 seconds, and rinse once with the color enhancement solution that comes with the kit; finally wash with water for 5 seconds, dry with filter paper or let it dry naturally, and observe under a microscope. If the staining is successful, seal the slides and use a digital slide scanner to capture images.

[0178] The results of H&E staining are as follows Figure 12 As shown, 4-week-old (4W) Prpf6 + / - There was no significant change in the morphology and retinal layers of mice and wild-type mice, but Prpf6 + / - The retinal thickness of mice was slightly different from that of wild-type mice ( Figure 12 , left). 5 months old (5M) Prpf6 + / - The retina of some mice and wild-type mice showed some damage, with changes in thickness and morphology, and loose connections between cells ( Figure 12 , right). This indicates that Prpf6 knockout leads to impaired morphology and function of the mouse retina.

[0179] The Prpf6 gene knockout mouse model obtained in the present invention, i.e., the RP disease model, has a slow phenotypic progression, which gradually develops over 1.5 months after birth and longer, allowing researchers to observe the progression and changes of the disease over a long period of time when using it.

[0180] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A Prpf6 A method for preparing a RP disease mouse model with haploinsufficiency caused by gene mutation, characterized in that: The method comprises: using the CRISPR / Cas9 system to Prpf6 Exons 4 to 11 of the gene are excised; the CRISPR / Cas9 system includes sgRNA and Cas9 enzyme, and the sgRNA sequences are shown in SEQ ID NOs. 1 to 4 respectively; The method specifically comprises the following steps: The sgRNA sequences shown in SEQ ID NO. 1 to 4 and the Cas9 protein were mixed to obtain an RNP injection complex, which was injected into mouse fertilized eggs, which were transplanted into surrogate mice, and mice were born and genotyped to obtain F0 generation mice with successful knockout. Prpf6 Gene mutations induce haploinsufficient RP in mice.

2. Use of the mouse model prepared by the method according to claim 1 in screening drugs for preventing or treating retinitis pigmentosa.

3. Use of the mouse model prepared by the method according to claim 1 in RP disease research, wherein the research is for the purpose of non-disease treatment.

4. Use of the mouse model obtained by the method of claim 1 in preparing drugs for identifying and / or testing retinitis pigmentosa.

5. The use according to claim 4, characterized in that The drug is used for preventing and / or treating retinitis pigmentosa and / or treating complications associated with retinitis pigmentosa.

6. A method for cultivating a mouse model of retinitis pigmentosa, characterized in that: The method comprises the following steps: mating the mouse models prepared by the method according to claim 1 with each other to obtain heterozygous mice.

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