Construction method and application of RP mouse model with insufficient haplodose caused by Prpf6 gene mutation

Abstract

The invention discloses a construction method and application of a Prpf6 gene mutation induced single dose insufficient RP mouse model, and belongs to the technical field of molecular biology and biomedicine. The mouse model constructed by using the method provides a convenient, reliable and economic means for researching the relationship between PRPF6 gene mutation and retinitis pigmentosa diseases and the pathogenesis of the PRPF6 gene mutation, and provides an effective research model capable of stably inheriting in the research on the aspects of RP pathogenesis, treatment methods, drug screening and the like. The animal model is subjected to transcriptome and a series of phenotype detection, wherein a transcriptome result shows that Prpf6 gene knockout mouse retina has occurred a certain degree of metabolic abnormality, electrophysiological detection, fundus examination and Hamp; e staining results show that the shapes and functions of the retinal cell layer and the retina of the mouse are damaged, and the disease phenotype of RP is met.

Description

Technical Field

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

[0002] CRISPR / Cas9 technology is a gene editing tool that has developed rapidly in recent years. It has high precision and efficiency and is widely used in the fields of genome editing, gene function research and genetic disease treatment. The CRISPR / Cas9 system was first discovered in bacteria as a bacterial immune system to fight against the invasion of foreign DNA. Its main components include sgRNA (single-guide RNA) and Cas9 nuclease. sgRNA guides Cas9 to a specific DNA target and destroys the target DNA through the double-strand cutting function of Cas9. The mechanism of gene knockout depends on the cell's repair of double-strand breaks, usually through non-homologous end joining (NHEJ) to produce base deletions or insertions, resulting in loss of function of the target gene. The high efficiency, simplicity and specificity of CRISPR / Cas9 make it perform well in the simultaneous editing of multiple gene sites. Despite the problems of off-target effects and repair efficiency, the technology has a wide range of applications including gene function research, disease model construction and gene therapy.

[0003] Retinitis Pigmentosa (RP) is a group of inherited retinal degenerative diseases characterized by progressive retinal photoreceptor cell apoptosis and retinal pigment epithelial degeneration, with obvious clinical and genetic heterogeneity. In 2018, RP was listed in the first batch of rare diseases announced in my country. The initial clinical manifestations of RP are night blindness and narrow vision, usually starting from adolescence. Then, with the irreversible degeneration of cones and rods, complete blindness eventually occurs. Currently available clinical drugs and methods cannot effectively delay or treat RP. Most RP pathogenic genes are specifically expressed in the retinal area and are involved in different functions of the photoreceptor system, such as phototransduction signals, retinal transcription factor-related pathways, retinal metabolism, cell structure, and ciliary structure and function. Among them, 6 small nuclear ribonucleoprotein particles (snRNP) components involved in splicing (PRPF3, PRPF4, PRPF6, PRPF8, PRPF31, SNRNP200) can also cause RP. In addition, we have previously collected more than 200 patients with retinitis pigmentosa and performed exome sequencing, and found multiple new snRNP gene mutations, including a new splicing site mutation of the PRPF6 gene (c.2673+1G>A). It is currently known that 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. There are too few studies on RP caused by PRPF6 mutations, and the molecular mechanism of RP caused by PRPF6 mutations still needs further exploration.

[0004] The PRPF6 gene (Pre-mRNA Processing Factor 6) encodes a spliceosome-related protein that participates 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 RP-related symptoms 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 comprising: removing the 4th to 11th exons of the Prpf6 gene in the mouse homologous chromosome without affecting the upstream and downstream genes, forming a deletion of about 17 kb in length, with a coding sequence of 1165 bp, accounting for 41.27% of CDS, and the knocked-out coding sequence is not a multiple of 3, resulting in a frameshift mutation, so that the function of the gene is destroyed, thereby achieving the knockout of Prpf6.

[0007] The present invention provides a method for preparing a non-human mammal model of retinitis pigmentosa (RP) disease, the method comprising: 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), which is a complete mature mRNA that has been sheared and processed; it can also refer to the sequence with Ensembl ID ENSMUSG00000002455; it can also refer to the transcript of Prpf6201: ENSMUST00000002529.

[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 transferring 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 a pseudo-pregnant female animal to produce F0 generation, 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 NO.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 NO. 1 to 4 in 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, mixing and incubating for 10 minutes to obtain the RNP injection complex.

[0022] The present invention also provides a mouse model of RP disease caused by haploinsufficiency due to Prpf6 gene mutation. The mouse model is prepared by removing exons 4 to 11 of the Prpf6 gene in the homologous chromosome of the mouse, 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 haploinsufficient induced by Prpf6 gene mutation, the method comprising: using the CRISPR / Cas9 system to remove 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 NO. 1 to 4 and the Cas9 protein are mixed to obtain an RNP injection complex, the RNP injection complex is injected into a mouse fertilized egg, the fertilized egg is transplanted into a surrogate mouse, mice are produced and genotyped, and F0 generation mice with successful gene knockout, i.e., RP disease mice, are obtained;

[0029] The F0 generation mice with successful gene knockout were crossed with wild-type mice to obtain the 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, that is, 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 one embodiment of the present invention, the sgRNA and Cas9 protein are added in a volume ratio of 7:1.

[0034] In one embodiment of the present invention, the sgRNA is obtained by mixing sgRNAs with sequences of SEQ ID NOs. 1 to 4 in a ratio of 1:1:1:1 (v / v / v / v).

[0035] 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 NO. 1 to 4 in 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, mixing and incubating for 10 minutes to obtain the RNP injection complex.

[0036] The present invention also provides a method for preparing a Prpf6 gene knockout mouse model, the method comprising: using the CRISPR / Cas9 system to remove exons 4 to 11 of the mouse Prpf6 gene.

[0037] 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.

[0038] In one embodiment of the present invention, the method specifically comprises the following steps:

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

[0040] The F0 generation mice with successful gene knockout were crossed with wild-type mice to obtain F1 generation heterozygous gene knockout mice;

[0041] The obtained F1 generation heterozygous male and female mice were mated with each other to obtain F2 generation heterozygous gene knockout mice;

[0042] The obtained F2 generation heterozygous male and female mice were mated with each other to obtain the Prpf6 gene knockout heterozygous mouse strain.

[0043] 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.

[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 NO.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 NO. 1 to 4 in 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, mixing and incubating for 10 minutes to obtain the 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 application of the Prpf6 gene knockout mouse model in studying the pathogenic mechanism of RP.

[0049] The present invention also provides application 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 in 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 aimed at 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 for preventing and / or treating retinitis pigmentosa, and / or treating 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 Prpf6 gene mutation-induced haploinsufficient RP disease mouse model or the mouse model prepared by the above-mentioned method with each other 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 by 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 where 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 PRPF6 mutation-related retinitis pigmentosa.

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

[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 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 constructing a 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 for gene identification of F0 generation mice.

[0067] Figure 4 This is the sequencing result diagram of 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 transcriptome sequencing of Prpf6 gene knockout mouse model.

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

[0073] Fig.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] Fig.11 For wild type and Prpf6 + / - Fundus photographs of mice; the left picture is a wild-type mouse fundus photograph; the right picture is a Prpf6 + / - Fundus photography of mice.

[0075] Fig.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 the excision of exons 4 to 11 of the mouse Prpf6 gene by non-homologous end joining and induce 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 the 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, sgRNA targeting the upstream region of exon 4 and the downstream region of exon 11 of the mouse Prpf6 gene was first synthesized, and then co-injected with Cas9 protein into mouse fertilized eggs to produce targeted knockout offspring F0. The genotype of F0 animals was identified by PCR and sequence analysis, and finally they were co-raised with wild-type mice to detect germline transmission and generate heterozygous animals to construct a mouse model of retinitis pigmentosa.

[0081] The specific construction process is as follows:

[0082] 1. Design of gene knockout target sites

[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 the mouse. The gene has 21 exons. The first exon contains an ATG start codon and the 21st exon contains a TGA stop codon. The transcript is Prpf6201: ENSMUST00000002529 transcript, and the knocked-out 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 higher scores near the upstream sequence of exon 4 and the downstream sequence of exon 11 of the mouse Prpf6 gene to design sgRNAs. The designed sgRNA sequence information is shown in SEQ ID NO. 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 horizontal bar indicates 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) complex

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

[0101] Then, 1.4 μL of 100 pmol / μL mixed sgRNA (obtained by mixing the sgRNA solutions of SEQ ID NO. 1 to 4 in 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 number: M0646M) was added, mixed and incubated for 10 minutes to obtain an RNP injection complex.

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

[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. Whether the female mice had mated was determined by checking 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, catalog number: M7292), and place it in a 37°C constant temperature 5% CO2 incubator and culture it to the 2-cell stage 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 mouse on the day of thrombus formation;

[0107] After the fallopian tube 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: 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] The genomic DNA of F0 mice was extracted by alkaline lysis solution. First, the tail tip tissue of 2-week-old F0 mice was retained, and 100 μL of solution A was added to a 1.5 mL EP tube, and then incubated at 100 ° C in a dry thermostat for 30 minutes. Finally, 100 μL of solution B was added, and the mixture was fully shaken and mixed, and centrifuged to obtain the genomic DNA of F0 mice and put into a 4 ° C refrigerator for use. The components of solution A are 25 mmol / L NaOH and 2 mmol / L EDTA; the components of solution B are 40 mmol / L Tris·HCl, and the pH value is 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 According to the PCR standard system, 25 μL of PCR mixture was prepared, 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 above F0 generation mouse genomic DNA;

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

[0117] The 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 17942 bp 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, mice with successful Prpf6 gene knockout were obtained. Figure 4 shown.

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

[0133] Examples of breeding routes include Figure 6 As shown. Selfing of (+ / -) heterozygous mice can produce (- / -) homozygous mice, (+ / -) heterozygous mice, and (WT) wild-type mice. 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] When the target band (865 bp fragment) was amplified by PCR on mouse DNA using primer set 2, it was determined that the target gene was not successfully deleted.

[0142] The genotypes of (- / -) homozygous, (+ / -) heterozygous, and (+ / +) wild-type mice were determined using primers F1, R1, and F2. The amplification results are as follows:

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

[0144] (+ / -) The target bands of heterozygous genotype were 668 bp and 865 bp;

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

[0146] Due to the importance of the Prpf6 gene to the growth and development of mice, the present invention found that it was impossible to breed homozygous Prpf6 knockout mice in subsequent breeding, and it was speculated that the embryos of homozygous Prpf6 knockout mice could not survive. Heterozygous genotype mice are Prpf6 knockout mice (Prpf6 + / - mice).

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

[0148] The F0 generation male mice with successful knockout of the target gene were crossed with wild-type female mice and caged. After 1 week, the pregnant female mice were raised in a single cage and gave birth to obtain F1 generation mice. Three weeks after the birth of the F1 generation mice, the tails were cut to extract tissue DNA, and the Prpf6 gene knockout mice were screened by PCR reaction and agarose gel electrophoresis detection. The specific operation was the same as the identification method of the F0 generation gene knockout mice, the primers used were shown in Table 3, and the PCR reaction conditions were shown in Table 4.

[0149] PCR identification results Figure 7 As shown, heterozygous Prpf6 knockout mice were obtained (Prpf6 + / - mice).

[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 and heterozygous mutant male mice obtained in the above step (1) were caged together. After 1 week, the pregnant female mice were caged separately and the F2 generation hybrid mice were obtained after delivery. Three weeks after the birth of the F2 generation hybrid mice, the tails were cut to extract tissue DNA, and the Prpf6 gene knockout mice were screened out by PCR reaction and agarose gel electrophoresis detection. The specific operation was the same as the F1 generation gene knockout mouse genotype identification method in the above step (1), 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] Female and male mice with knockout heterozygous were selected from the F2 generation mutants. After being caged together for 1 week, the pregnant female mice were raised in a single cage and the F3 generation mice were obtained after delivery. Three weeks after the birth of the mice, one mouse was selected from each litter to extract tissue DNA by tail clipping. The genotype was identified by PCR amplification and agarose gel electrophoresis to screen out Prpf6 knockout mice. The specific operation was the same as the above-mentioned method for identifying the genotype of the knockout mice. The primers used were shown in Table 3, and the PCR reaction conditions were shown in Table 4.

[0158] The obtained F3 generation heterozygous mice were housed together, bred and expanded, and finally 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 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. The retinas were then detached using ophthalmic scissors and ophthalmic forceps under a stereomicroscope.

[0161] Use 1mL Trizol reagent purchased from Kangwei Century to digest the retinal tissue at room temperature. After the tissue is completely digested, add 200μL of chloroform and mix well, then centrifuge at 4℃, 1200rpm for 15min, transfer 500μL of the supernatant after centrifugation to a new 1.5mL EP tube without RNase enzyme, and then add 500μL of isopropanol to precipitate RNA, mix well, centrifuge at 4℃, 1200rpm for 5min, carefully remove the supernatant after centrifugation, add 1mL 75% ethanol to wash the precipitate, centrifuge at 4℃, 1200rpm for 5min, carefully remove the supernatant after centrifugation, and finally dry at room temperature for 10min, and add 30μL of RNase enzyme-free water to dissolve RNA.

[0162] The obtained RNA was sent to Beijing Novogene Co., Ltd. for transcriptome sequencing, and the sequencing results were subjected to transcriptomic analysis, and 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 figure, 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, and the inflammatory-related leukocyte transendothelial migration pathway ( Fig. 9 ). The functions and pathways mentioned above are closely related to the retina, indicating that the retina of Prpf6 knockout mice has undergone a certain degree of metabolic abnormalities, which verifies the Prpf6 knockout mouse model to a certain extent.

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

[0165] (1) Mouse ophthalmological electrophysiological (ERG) examination

[0166] Five-month-old wild-type mice and Prpf6 knockout mice (Prpf6 + / - Five mice were gently taken out of the cage and sent to Beijing Xinlian Optoelectronics for ERG examination. Avertin (tribromoethanol (Mei Lun Biotechnology Co., Ltd.) was used to anesthetize the mice, and the anesthetic dosage was calculated according to the standard 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 timing 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; there was no obvious change in the b-wave time of rods and cones in the mouse retina.

[0171] The results showed that the peak amplitudes of both a-wave and b-wave decreased significantly. Fig.10 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. Fundus photography of the mice was continued. Anesthesia and mydriasis 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 ( Fig.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. They were perfused through the left ventricle with 30 mL of normal saline, and then the eyeballs of the mice were removed and fixed in a modified Davidson's fixative (A) purchased by Regen Biotechnology. 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 4μm 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, use a non-toxic and environmentally friendly dewaxing agent to dewax the paraffin sections, 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 warm water for 2 minutes; take them out of the warm water and shake off excess water, so that the distillation can evenly wet the entire tissue, put them in the nuclear stain hematoxylin for about 5 minutes, and wash with water for 5 seconds; then put them in the pulp stain eosin for about 15 seconds, rinse once with the color enhancement solution that comes with the kit; finally wash with water for 5 seconds, dry with filter paper or dry naturally, observe under a microscope, and if the staining is successful, seal the slides and use a digital slide scanner to collect images.

[0178] The results of H&E staining are as follows Fig.12 As shown, 4-week-old (4W) Prpf6 + / - There was no significant change in the morphology and retinal layers of the retina between mice and wild-type mice, but Prpf6 + / - The retinal thickness of mice was slightly changed compared with that of wild-type mice ( Fig.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 ( Fig.12 , right). This indicates that Prpf6 knockout leads to impaired morphology and function of the retina in mice.

[0179] The Prpf6 gene knockout mouse model obtained by the present invention, i.e., the RP disease model, has a slow phenotype progression, which gradually develops within 1.5 months after birth and longer, so that researchers can observe the progression and changes of the disease for a long time when using it.

[0180] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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 mouse model of RP disease caused by haploinsufficiency of Prpf6 gene mutation, characterized in that: The mouse model is prepared by removing exons 4 to 11 of the Prpf6 gene in the homologous chromosome of the mouse to achieve knockout of Prpf6.

2. A method for preparing a mouse model of RP disease caused by haploinsufficiency due to Prpf6 gene mutation, characterized in that: The method comprises: using the CRISPR / Cas9 system to excise exons 4 to 11 of the mouse Prpf6 gene; The CRISPR / Cas9 system includes sgRNA and Cas9 enzyme, and the sgRNA sequences are shown in SEQ ID NOs. 1 to 4, respectively.

3. The preparation method according to claim 2, characterized in that: 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, and the fertilized eggs were transplanted into surrogate mice. The mice were produced and their genotypes were identified to obtain successfully knocked out F0 generation mice, i.e., RP disease mice with haploinsufficient Prpf6 gene mutation.

4. A method for preparing a Prpf6 gene knockout mouse model, characterized in that: The method comprises: using the CRISPR / Cas9 system to excise exons 4 to 11 of the mouse Prpf6 gene; the CRISPR / Cas9 system comprises sgRNA and Cas9 enzyme, and the sgRNA sequences are shown in SEQ ID NOs. 1 to 4, respectively.

5. The preparation method according to claim 4, characterized in that: 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 a mouse fertilized egg, which was transplanted into a surrogate mouse, and mice were produced and genotyped to obtain F0 generation mice with successful Prpf6 knockout.

6. Use of the RP disease mouse model with haploinsufficiency caused by Prpf6 gene mutation as described in claim 1 or the mouse model prepared by any method described in claims 2 to 5 in screening drugs for preventing or treating retinitis pigmentosa.

7. Use of the RP disease mouse model with haploinsufficiency caused by Prpf6 gene mutation as described in claim 1 or the mouse model prepared by any of the methods described in claims 2 to 5 in RP disease research, wherein the research is for the purpose of non-disease treatment.

8. Use of the mouse model according to claim 1 or the mouse model obtained by the method according to any one of claims 2 to 5 in the preparation of a drug for identifying and / or testing retinitis pigmentosa.

9. The use according to claim 8, characterized in that: The drug is used for preventing and / or treating retinitis pigmentosa and / or treating complications associated with retinitis pigmentosa.

10. A method for cultivating a mouse model of retinitis pigmentosa, characterized in that: The method comprises the following steps: mating the RP disease mouse model caused by haploinsufficiency caused by Prpf6 gene mutation described in claim 1 or the mouse model prepared by any one of the methods described in claims 2 to 5 to obtain heterozygous mice.

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