A method for constructing a mouse model with ubap1l gene knockout and successfully reproducing a retinal pigment degeneration phenotype and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a construction method and application of a mouse model with Ubap1L gene knockout and successfully reproduced retinal pigment degeneration phenotype. The method comprises the following steps: designing sgRNA according to Ubap1L gene; the sgRNA comprises sgRNA1 as shown in SEQ ID NO: 5 and sgRNA2 as shown in SEQ ID NO: 6; mixing sgRNA and Cas9 protein to obtain gene editing liquid; transferring the gene editing liquid into mouse zygotes; transplanting the zygotes into pseudopregnant mice to obtain F0 generation mice; screening homozygous offspring of Ubap1L gene based on the F0 generation mice to obtain homozygous mice with Ubap1L gene knockout. The model successfully reproduces the typical pathological phenotype of human retinal pigment degeneration, has the characteristics of genetic stability and reliable phenotype, and provides an effective animal model for studying retinal degeneration mechanism, drug screening and gene therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for constructing and applying a mouse model of retinitis pigmentosa phenotype with UBAP1L gene knockout and successful replication. Background Technology

[0002] Retinitis Pigmentosa (RP) is a common inherited retinal disease with a global incidence of approximately 1 in 4000. It is one of the leading causes of irreversible vision loss and blindness in working-age individuals and adolescents. The typical pathological feature of the disease is the progressive damage and degeneration of photoreceptor cells (including rod and cone cells), accompanied by the gradual thinning and even disappearance of the outer nuclear layer of the retina. Corresponding pathological changes also occur in the retinal pigment epithelium and adjacent supporting cells. Patients often present with night blindness and progressive narrowing of the visual field. As the disease progresses, central vision may severely decline, eventually leading to complete blindness. RP exhibits high genetic heterogeneity, with over 100 related pathogenic genes identified. Inheritance patterns include autosomal dominant, autosomal recessive, and X-linked inheritance, and its molecular mechanisms are complex and diverse. This diversity in genetics and mechanisms not only results in a lack of systematic explanation of the disease mechanisms of RP but also poses a significant challenge to developing effective treatment strategies.

[0003] Currently, existing models have limitations such as their inability to mimic the human retinitis pigmentosa phenotype, which restricts in-depth exploration of the pathogenesis of retinitis pigmentosa and efficient screening of targeted drugs. Therefore, constructing a novel animal model that can stably mimic the typical characteristics of retinitis pigmentosa is of urgent and significant scientific and clinical value for revealing the molecular mechanisms of the disease, advancing research on its pathogenesis, and screening drugs with therapeutic potential. Summary of the Invention

[0004] Based on this, the present invention addresses the technical deficiency of existing Ubap1l gene knockout animal models in failing to reproduce the retinitis pigmentosa phenotype, and provides a novel method for constructing and applying a Ubap1l gene knockout mouse model. This model precisely knocks out the genomic region covering exons 3 to 6 using CRISPR-Cas9 technology, and for the first time successfully reproduces in mice a phenotype of progressive photocell degeneration and visual function decline similar to that of human RP patients.

[0005] The present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for constructing an Ubap1l gene knockout mouse model, comprising the following steps:

[0007] sgRNAs were designed based on the Ubap1l gene; the sgRNAs include sgRNA1 as shown in SEQ ID NO: 5 and sgRNA2 as shown in SEQ ID NO: 6;

[0008] The sgRNA and Cas9 protein were mixed to obtain the gene editing solution;

[0009] The gene-editing solution was transferred into mouse zygotes, and the zygotes transferred into the gene-editing solution were transplanted into pseudopregnant mice to obtain F0 generation mice;

[0010] Homozygous offspring of the Ubap1l gene were screened from F0 generation mice to obtain homozygous mice with Ubap1l gene knockout.

[0011] Furthermore, the sgRNA-targeted knockout region covers the splicing sites of exons 3 to 6 of the Ubap1l gene and the coding region of the UBA2 domain; the sgRNA-targeted knockout region is located on mouse chromosome 9, with NCBI accession number: GenBank: NM_001111145.2, specific location information g. 65277569-65285931, and a length of 8323 bp.

[0012] Furthermore, the sgRNA1 and sgRNA2 are mixed in a molar ratio of 1:1 to 2.

[0013] Furthermore, the Cas9 protein and sgRNA for targeting the mouse Ubap1l gene are mixed in a molar ratio of 1 to 2:1 in the gene editing solution.

[0014] Furthermore, the knockout region includes the SOUBA structural domain and the adjacent UMA-SOUBA connection region.

[0015] Furthermore, the gene-editing solution is transferred into mouse zygotes via microinjection.

[0016] Furthermore, methods for screening homozygous progeny of the Ubap1l gene based on F0 generation mice include:

[0017] F0 generation mice with positive germline transmission were backcrossed with wild-type mice to obtain F1 generation heterozygous mice.

[0018] The F1 generation heterozygous mice were crossbred to obtain the F2 generation homozygous mice, which are the Ubap1l gene knockout mouse models.

[0019] Furthermore, the mouse strain is C57BL / 6JGpt mouse.

[0020] C57BL / 6JGpt inbred mice (SPF grade) were selected. C57BL / 6JGpt mice have a clear genetic background and stable retinal phenotype, with low baseline levels of photoreceptor degeneration, which can minimize the interference of genetic variations on Ubap1l mutation-related phenotypes and ensure the reproducibility of experimental data. Furthermore, this strain exhibits high CRISPR / Cas9 editing efficiency and excellent germline passaging stability, making it suitable for constructing a heritable Ubap1l gene defect model.

[0021] Secondly, the present invention includes the application of the Ubap1l gene knockout mouse model constructed by the preparation method described above in screening drugs for the treatment of hereditary retinal diseases.

[0022] Furthermore, the hereditary retinal disease is retinitis pigmentosa.

[0023] Furthermore, the retinitis pigmentosa phenotype shown is characterized by progressive degeneration of photoreceptor cells and decline in visual function.

[0024] Thirdly, the present invention includes a Ubap1l Gene knockout kit, including: sgRNA-1, sgRNA-2 and Cas9 proteins;

[0025] The sequence of sgRNA-1 is shown as 5'-TGGGTGTCACAGGCAACCTC-3', and the sequence of sgRNA-2 is shown as 5'-AACGTTCTTGGGTTCACTCT-3'.

[0026] Among them, sgRNA-1 and sgRNA-2 are sgRNAs designed based on the knockout region of the Ubap1l gene;

[0027] The sgRNA-targeted knockout region covers the splicing sites of exons 3 to 6 and the coding region of the UBA2 domain; the sgRNA-targeted knockout region is located on mouse chromosome 9, NCBI accession number: GenBank: NM_001111145.2, specific location information g. 65277569-65285931, and length 8323 bp.

[0028] The present invention has the following beneficial effects:

[0029] 1. This invention utilizes CRISPR-Cas9 gene editing technology. By designing a sgRNA specifically targeting the Ubap1l gene and forming a gene editing solution with Cas9 protein, a stably heritable Ubap1l gene knockout mouse model was successfully constructed via microinjection and embryo transfer. This model successfully reproduces, for the first time, the core pathological features of retinitis pigmentosa (RP) patients, including progressive degeneration of photoreceptor cells and visual function decline. The phenotype is well-defined and genetically stable, solving the key problem of lacking ideal animal models for RP in existing technologies. This provides a reliable in vivo tool for in-depth research on the pathogenesis of RP, drug screening, and the development of treatment strategies.

[0030] 2. In this invention, a frameshift mutation and complete gene knockout are effectively achieved by precisely targeting and deleting an 8323 bp genomic fragment containing exon 3 to 6 splicing sites and the coding region of the UBA2 domain.

[0031] 3. The knockout region selected in this invention simultaneously covers the SOUBA domain and its adjacent UMA-SOUBA linker region, which not only achieves gene knockout but also reveals the synergistic role of this domain in maintaining protein function and retinal homeostasis.

[0032] 4. This model provides a reliable in vivo evaluation model for screening targeted drugs and gene therapy for the treatment of retinitis pigmentosa, as well as for studying the regulatory mechanism of retinitis pigmentosa, and has important translational application value. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is an expression profile of the UBAP1L / Ubap1l gene in various tissues of humans and mice.

[0035] Figure 2 Maps showing the localization of the UBAP1L / Ubap1l gene in the human and mouse retinas; Figure 2 A in the image is the signal map of the human retinal UBAP1L probe (20×). Figure 2 B is a magnified view of the UBAP1L probe signal in the human retina (63×). Figure 2 C in the figure is a marker of rhodopsin in the human retina (20×). Figure 2 D in the image is a magnified view of a localized rhodopsin marker in the human retina (63×). Figure 2E in the diagram is a human retinal RNAscope merged channel map (20×). Figure 2 F in the image is a magnified view of the combined channel of the human retinal RNAscope (63×). Figure 2 G in the image represents the mouse retinal Ubap1l probe signal (20×). Figure 2 H in the image is a magnified view (63×) of the Ubap1l probe signal in the mouse retina. Figure 2 In the diagram, I represents the rhodopsin protein marker in the mouse retina (20×). Figure 2 J in the image is a magnified view of the rhodopsin protein labeling in the mouse retina (63×). Figure 2 K in the figure represents the mouse retinal RNAscope merged channel map (20×). Figure 2 The L in the image is a magnified view of the combined channel of the mouse retinal RNAscope (63×).

[0036] Figure 3 This is a schematic diagram illustrating the construction process of the Ubap1l gene knockout mouse model.

[0037] Figure 4 This is a diagram showing the Sanger sequencing results of wild-type mice and gene knockout mice used in this invention.

[0038] Figure 5 This is a graph showing the results of mRNA expression level detection in Ubap1l gene knockout mice in this invention.

[0039] Figure 6 Ubap1l gene knockout mice (Ubap1l) ​​were 3 months old. - / - ) and wild-type mice (Ubap1l) + / + Retinal fundus photography and retinal potential (ERG) map; among which, Figure 6 A in the text is Ubap1l + / + Fundus image of the right retina of a mouse; Figure 6 B in the text is Ubap1l + / + Fundus image of the left retina of a mouse; Figure 6 C in the text is Ubap1l - / - Fundus image of the right retina of a mouse; Figure 6 D in the text refers to Ubap1l - / - Fundus image of the left retina of a mouse; Figure 6 E in the text is Ubap1l + / + Raw ERG waveforms of mice under dark adaptation; Figure 6 G in the context is the same as Ubap1l. - / - Raw ERG waveforms of mice under dark adaptation; Figure 6 F in the figure represents the b-wave amplitude-light intensity curve of the scotopic ERG. Figure 6H in the figure represents the b-wave amplitude-light intensity curve of the photoluminescent ERG.

[0040] Figure 7 Ubap1l gene knockout mice (Ubap1l) ​​were 6 months old. - / - ) and wild-type mice (Ubap1l) + / + Retinal fundus photography and retinal potential (ERG) map; among which, Figure 7 A in the text is Ubap1l + / + Fundus image of the right retina of a mouse; Figure 7 B in the text is Ubap1l + / + Fundus image of the left retina of a mouse; Figure 7 C in the text is Ubap1l - / - Fundus image of the right retina of a mouse; Figure 7 D in the text refers to Ubap1l - / - Fundus image of the left retina of a mouse; Figure 7 E in the text is Ubap1l + / + Raw ERG waveforms of mice under dark adaptation; Figure 7 F in the figure represents the b-wave amplitude-light intensity curve of the scotopic ERG. Figure 7 G in the context is the same as Ubap1l. - / - Raw ERG waveforms of mice under dark adaptation; Figure 7 H in the figure represents the b-wave amplitude-light intensity curve of the photoluminescent ERG.

[0041] Figure 8 Ubap1l gene knockout mice (Ubap1l) ​​were 9 months old. - / - ) and wild-type mice (Ubap1l) + / + Retinal fundus photography and retinal potential (ERG) map; among which, Figure 8 A in the text is Ubap1l + / + Fundus image of the right retina of a mouse; Figure 8 B in the text is Ubap1l + / + Fundus image of the left retina of a mouse; Figure 8 C in the text is Ubap1l - / - Fundus image of the right retina of a mouse; Figure 8 D in the text refers to Ubap1l - / - Fundus image of the left retina of a mouse; Figure 8 E in the text is Ubap1l + / + Raw ERG waveforms of mice under dark adaptation; Figure 8 F in the figure represents the b-wave amplitude-light intensity curve of the scotopic ERG. Figure 8 G in the context is the same as Ubap1l. - / - Raw ERG waveforms of mice under dark adaptation; Figure 8H in the figure represents the b-wave amplitude-light intensity curve of the photoluminescent ERG.

[0042] Figure 9 Ubap1l for different ages + / + With Ubap1l - / - Morphological comparison of rod photoreceptors in mice (n=3); Figure 9 A in the text represents 3-month-old Ubap1l + / + Morphological diagram of rod photoreceptors in the mouse retina; Figure 9 B in the text refers to 3-month-old Ubap1l + / + A magnified view of the morphology of the rod photoreceptors in the mouse retina; Figure 9 C in the text represents 6-month-old Ubap1l + / + Morphological diagram of rod photoreceptors in the mouse retina; Figure 9 D in the text refers to 6-month-old Ubap1l + / + A magnified view of the morphology of the rod photoreceptors in the mouse retina; Figure 9 E in the text refers to 9-month-old Ubap1l + / + Morphological diagram of rod photoreceptors in the mouse retina; Figure 9 F in the text refers to 9-month-old wild-type mice, Ubap1l. + / + A magnified view of the morphology of the rod photoreceptors in the mouse retina; Figure 9 G in the text refers to 3-month-old Ubap1l - / - Morphological diagram of rod photoreceptors in the mouse retina; Figure 9 H in the text refers to Ubap1l at 3 months of age. - / - A magnified view of the morphology of the rod photoreceptors in the mouse retina; Figure 9 The I in the text refers to 6-month-old Ubap1l - / - Morphological diagram of rod photoreceptors in the mouse retina; Figure 9 J in the text refers to 6-month-old Ubap1l - / - A magnified view of the morphology of the rod photoreceptors in the mouse retina; Figure 9 K in the text refers to Ubap1l at 9 months of age. - / - Morphological diagram of rod photoreceptors in the mouse retina; Figure 9 L in the text refers to 9-month-old Ubap1l - / - A magnified view of the morphology of the rod photoreceptors in the mouse retina. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0044] Example 1: Validation of the expression level and location of UBAP1L / Ubap1l in human / mouse model retinal tissue

[0045] 1. Experimental Methods

[0046] 1.1 Sample Source

[0047] Human samples: RNA expression profiling was performed on 29 different anatomically distinct tissues. Six ocular tissues (retina, cornea, lens, choroid, iris, and ciliary body) were obtained from donor eyes (isolated under RNase-free conditions); the other 23 extraocular samples were purchased from Clontech Laboratories in the United States and were derived from 12 Caucasian individuals aged 18-54 who died suddenly.

[0048] Mouse samples: Wild-type C57BL / 6J mice were selected, and nine types of ocular tissues (retina, choroid, cornea, lens, iris, ciliary body, optic nerve, sclera, and extraocular muscles) and 21 types of extraocular tissues were collected to analyze the RNA expression profile of the mouse Ubap1l gene.

[0049] 1.2 Experimental Methods

[0050] 1.2.1 Human-mouse RNA expression profile analysis

[0051] The expression profiles of the UBAP1L / Ubap1l gene in various tissues were systematically analyzed using real-time quantitative PCR (qPCR). Total RNA from human eye tissue and all mouse tissues was extracted using TRIzol reagent (Invitrogen, USA). RNA concentration and purity were determined using a NanoDrop spectrophotometer. Total RNA was reverse transcribed into cDNA using a PrimeScript™ reverse transcription kit (RR047A, TaKaRa, Japan). Amplification was performed using PowerUp SYBR Green premixed buffer (A25742, Applied Biosystems, USA) on a QuantStudio™ Dx real-time quantitative PCR instrument. Relative quantification of RNA levels was performed using the ΔΔCt method, with GAPDH as an internal reference gene. The qPCR primers designed using Primer 3 software (http: / / primer3.ut.ee / ) are shown below:

[0052] Table 1. Primer sequences for qPCR validation of UBAP1L / Ubap1l gene mRNA expression profile

[0053]

[0054] 1.2.2 Spatiotemporal localization of UBAP1L transcripts in human and mouse retina (RNAscope in situ hybridization)

[0055] Spatial localization analysis of UBAP1L / Ubap1l mRNA in paraffin-embedded human and mouse retinal sections was performed using RNAscope multiplex fluorescence in situ hybridization. The specific method was as follows: After dewaxing, paraffin-embedded sections were pretreated with hydrogen peroxide, followed by antigen retrieval in 95°C antigen retrieval buffer for 15 minutes. Hydrophobic boundaries were drawn around the tissue using an ImmEdge hydrophobic barrier pen (ACD, catalog number: 310018), and the sections were placed in a humidified chamber and treated with proteinase K at 40°C for 30 minutes. After rinsing, the UBAP1L mRNA probe / Ubap1l mRNA probe was hybridized at 40°C for 2 hours, followed by multiple rounds of amplification (30 minutes each). Signal amplification was achieved using TSA Vivid fluorescein 570 reagent. RNAscope-stained slides were further co-stained with rhodopsin antibody (Abcam, Ab5417), opsin antibody (Merck Millipore, AB5405), and RPE65 antibody (Abcam, Ab231782-10) to confirm the specific location of the Ubap1l transcript. High-resolution imaging analysis was performed using a Zeiss LSM980 confocal microscope system with 20× or 63× objectives (1.0× zoom) controlled by Zen Blue acquisition software.

[0056] 2. Experimental Results

[0057] like Figure 1 As shown, the UBAP1L / Ubap1l gene is specifically highly expressed in human and mouse retinal tissues, but exhibits relatively low gene expression in both human and mouse central nervous system tissues. Figure 2 The AF study, using RNAscope technology for precise localization, revealed that human UBAP1L mRNA is specifically enriched in the outer nuclear layer (ONL) and inner segment (IS) regions of photoreceptor cells, and the UBAP1L signal co-localized with rhodopsin antibody overlaps with photoreceptor markers. Only a weak signal was observed in the retinal pigment epithelium (RPE) layer. Figure 2 The GL results show that mouse Ubap1l mRNA is also confined to the outer nuclear layer and inner segments, and its expression pattern is highly consistent with that of humans. In summary, this invention, for the first time, reveals the localized expression of the UBAP1L / Ubap1l gene in the human and mouse retinas using RNAScope technology. Previously, the precise localization of UBAP1L in the retina had remained unclear (due to non-specific staining results from Ubap1l-customized antibody immunofluorescence staining), indicating that the UBAP1L / Ubap1l gene plays an important role in the retina.

[0058] Example 2: Construction and Identification of Ubap1l Gene Knockout Mouse Model

[0059] 1. Laboratory animals

[0060] Wild-type C57BL / 6 mice, SPF grade, 6 weeks old, purchased from Jiangsu Jicui Biotechnology Co., Ltd.

[0061] 2. Construction of Ubap1l gene knockout mouse model

[0062] Based on the Ensembl database (ENSMUST00000147185.2), the mouse Ubap1l-201 transcript was selected as the target gene. This gene is located on mouse chromosome 9 (Chr9:65268343-65287659), and its exon structure and coding region are located in the CDS: 65276503-65287561 interval. sgRNA1 and sgRNA2 were designed to be located at intron 2 and intron 6 of the Ubap1l gene, respectively. The knockout region targets introns 2 to 6 (GenBank: NM_001111145.2, g.65277569-65285931), which covers the splice sites of exons 3-6 and the coding region of the UBA2 domain. The gene sequences of sgRNA1 and sgRNA2 are shown in Table 2 as SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0063] Table 2 Sequence listings of sgRNA1 and sgRNA2

[0064]

[0065] like Figure 3 As shown, a mouse model of Ubap1l gene knockout was constructed using CRISPR / Cas9 gene editing technology. The specific method includes the following steps:

[0066] S1, based on the knockout region, specifically targets sgRNA1 and sgRNA2;

[0067] S2, active sgRNA1, sgRNA2 and Cas9 protein were microinjected into C57BL / 6JGpt mouse zygotes in a molar ratio of 1:1:1, with a total injection volume of 1 nL. The zygotes were then transplanted into pseudopregnant female mice, and F0 generation mice were obtained after the mice became pregnant and gave birth.

[0068] S3, select the F0 generation mice with positive germline transmission obtained in step S2 and backcross them with C57BL / 6JGpt wild-type mice to obtain F1 generation heterozygous mice;

[0069] S4. Cross the F1 generation heterozygous mice obtained in step S3 to obtain the F2 generation homozygous mice, which are the Ubap1l gene knockout mouse models.

[0070] 3. Validation of the Ubap1l gene knockout mouse model

[0071] 3.1 Genotyping

[0072] The primer sequences used to identify the genotype of the mouse model were designed according to the construction strategy. Two pairs of primers were designed to amplify mouse genomic DNA by PCR to identify whether the Ubap1l gene was successfully knocked out. The primer sequences are shown in Table 3.

[0073] Approximately 2-3 mm of tail tip tissue was excised from mice aged 14-21 days after birth. Lysis buffer containing protease was added, and the mixture was incubated overnight at 55°C. The following day, after centrifugation, the supernatant was collected and an equal volume of isopropanol was added. The mixture was repeatedly inverted and mixed until white flocculent DNA precipitated. Centrifugation was repeated, and the supernatant was discarded. The mixture was washed twice with 75% ethanol and air-dried. The precipitate was dissolved in ddH2O or pure water and used as the DNA template for PCR amplification. PrimeSTAR Max Premix (Takara #R045A) was used for PCR. Mouse tail DNA was amplified using wild-type and knockout primers, respectively. The PCR reaction system is shown in Table 4. PCR products were subjected to agarose gel electrophoresis (using a 1.5% agarose gel, 120V for 30 minutes), and the bands were finally interpreted using an imaging system (Bio-Rad ChemiDoc).

[0074] Table 3 PCR Primer Sequence List

[0075]

[0076] Table 4. PCR reaction system for identifying Ubap1l knockout mice

[0077]

[0078] Further Sanger sequencing was performed. PCR amplification products from homozygous and wild-type mice were mixed with magnetic beads at a 1:1 ratio and repeatedly pounded to ensure complete binding. Centrifuge tubes were placed on a magnetic rack and allowed to stand until the liquid was clear and the magnetic beads were completely adsorbed onto the tube walls. The supernatant was carefully discarded while the centrifuge tubes remained on the magnetic rack. The tubes were washed twice with freshly prepared 70% ethanol and then dried at room temperature. Elution water was added, and the mixture was pounded to reconstitute the magnetic beads. The tubes were allowed to stand again to promote DNA dissociation from the beads. The centrifuge tubes were placed back on the magnetic rack and allowed to stand until the beads were completely adsorbed. The supernatant containing the purified DNA was transferred to a new centrifuge tube, which was the purified PCR product. The concentration of the purified DNA was measured using a Nanodrop micro-spectrophotometer. Agarose gel electrophoresis was performed on the purified product to confirm that the bands were single and undegraded. Using the purified magnetic bead product as a template, wild-type / knockout forward and reverse primers were used for Sanger sequencing reaction based on the BigDye Terminator kit. The sequencing primer sequences and reaction procedures are shown in Tables 5 and 6.

[0079] After the sequencing reaction is complete, any unincorporated ddNTPs, primers, and salt ions must be removed; otherwise, they will interfere with the capillary electrophoresis results of the 3500 sequencer. Add 10 μL of Hi-Di formamide (sequencing grade) to dissolve the DNA, denature at 95°C for 5 minutes, then immediately incubate on ice for 5 minutes before sequencing on the 3500 sequencer. After sequencing, obtain the raw sequencing peak image file and use sequence analysis software (such as SnapGene) to check the peak image quality (focusing on signal intensity, interference peaks, etc.), and compare it with the reference sequence for verification.

[0080] Table 5. Sanger sequencing primer sequences

[0081]

[0082] Table 6. Sanger reaction system for identifying Ubap1l knockout mice

[0083]

[0084] 3.2 Validation of mRNA expression silencing

[0085] Gene knockout at 3 months of age (Ubap1l) - / - ) and wild type (Ubap1l + / +Three mice (three of each type, six in total) were used, and their genotypes were confirmed to be correct through genotyping. The mice were injected intraperitoneally with 1% sodium pentobarbital, and their reactions were observed until there was no corneal reflex and no retraction when the toes were squeezed, confirming adequate anesthesia. The mice were then fixed supine on a dissecting board, and their abdomens were disinfected by wiping with 75% ethanol. The thoracic cavity was cut along the midline to expose the heart, and the right atrial appendage was cut open with ophthalmic scissors (as a reflux port). 10 mL of pre-cooled saline was placed in a syringe, connected to a 23G blunt-tipped perfusion needle, and slowly inserted into the left ventricle (needle tip towards the aorta). Saline was injected at a rate of 1-2 mL / min until the liver and limbs turned white, ensuring complete removal of circulating blood. Using ophthalmic forceps, grasp the base of the eyeball, sever the optic nerve, and quickly remove both eyeballs. Under an ice-cold dissecting microscope, use ophthalmic scissors to cut open the eyeball along the limbus, removing the lens and vitreous body. Gently lift the retinal edge with forceps and completely dissect the retinal tissue. Immediately place the separated retina into a 1.5 mL RNase-free centrifuge tube pre-filled with 1 mL Trizol. Use a pipette to repeatedly pipette until tissue lysis occurs, and allow to stand at room temperature to promote the dissociation of nucleoprotein complexes. Add chloroform at 1 / 5 the volume of Trizol for RNA extraction, centrifuge at 12000×g for 20 minutes at 4°C, and the solution will separate into layers (lower red organic phase containing protein, middle layer containing DNA, and upper colorless aqueous phase containing RNA). Carefully aspirate the upper aqueous phase (approximately 400-500 μL, avoiding contact with the middle layer) into a new RNase-free centrifuge tube; add an equal volume of isopropanol, gently invert to mix, and allow to stand at room temperature for 10 minutes; centrifuge at 12000×g for 20 minutes at 4°C, and a white RNA precipitate will be visible at the bottom of the tube. Discard the supernatant, add 1 mL of 75% ethanol (prepared with DEPC water), and gently invert to wash the precipitate; centrifuge at 7500×g for 10 minutes at 4℃, discard the ethanol (try to remove as much as possible); dry at room temperature; add DEPC water and vortex to dissolve the precipitate (adjust the volume according to the amount of RNA), and use Nanodrop to detect the RNA concentration and purity.

[0086] Following the instructions of the PrimeScript™ RT Reagent Kit, genomic DNA (gDNA) removal and reverse transcription were performed sequentially to obtain a cDNA template for later use. qPCR primers for the Ubap1l gene and Gadph internal reference gene were designed using the Primer 3 website. The qPCR primers spanned at least one intron, and the primer sequences are shown in Table 7.

[0087] Table 7. qPCR primer sequence list for Ubap1l gene and internal reference gene Gapdh

[0088]

[0089] The qPCR reaction system included 2×SYBR Premix Ex Taq II, forward and reverse primers, cDNA template, and water replenished with ddH2O. Biological replicates (3 mouse samples per group) and biophysical replicates were set up. The amplification program was run: 95℃ pre-denaturation for 30 seconds, followed by 40 cycles (95℃ denaturation for 5 seconds, 60℃ annealing extension for 30 seconds with fluorescence signal acquisition). Finally, melting curve analysis was performed (95℃ 15 seconds → 60℃ 1 minute → 95℃ 15 seconds). Primer specificity was verified by melting curve analysis, and the relative expression level of the target gene Ubap1l was calculated (ΔΔCt method) by combining the Ct value of the internal control Gapdh.

[0090] 3.3 Phenotypic Validation

[0091] 3.3.1 Electroretinography (ERG) Experiment

[0092] Gene knockout mice (Ubap1l) ​​at 3 months, 6 months, and 9 months of age were selected. - / - ) and its littermate background-matched wild-type mice (Ubap1l) + / + Each genotype group must contain at least 6 mice (male to female ratio of 1:1, excluding individuals with diseases or deformities). The mouse genotype must be confirmed by genotyping before the experiment. Dark adaptation (complete darkness, ≥12 hours) begins 24 hours prior to the experiment, with free access to food and water to ensure the retina is in a dark-vision state.

[0093] Weigh the mice and administer an intraperitoneal injection of 1% sodium amobarbital at a dose of 10 μl / g. Gently massage the abdomen after injection to promote absorption. Observe the anesthesia status until there is no corneal reflex and no retraction when the hind limbs are squeezed, confirming that the depth of anesthesia is appropriate (too shallow anesthesia may cause struggling and affect recording, while too deep anesthesia may inhibit nerve responses). Instill compound tropicamide eye drops into both eyes to ensure that the pupils are fully dilated (diameter ≥2 mm) and avoid iris obstruction affecting electrode contact with the cornea.

[0094] ERG was tested in a completely dark room environment (background light ≤ 0.01 cd / m²). 2 The procedure was performed using the CELERIS system (D430, Diagnostics LLC, USA), equipped with a stimulation electrode, a reference electrode (placed subcutaneously behind the ear), and a ground electrode (placed subcutaneously in the tail). Hydroxypropyl methylcellulose eye drops were used for electrode fixation. The mouse was placed supine on a warming table, and hydroxypropyl methylcellulose eye drops were applied to the corneal surface. The stimulation electrode was gently placed in the center of the cornea, ensuring full contact between the electrode and the cornea without air bubbles.

[0095] Stimulation parameter settings:

[0096] Dark-adapted ERG (Scotopic ERG): Under dark-adapted conditions, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 cd·s / m were applied sequentially. 2 Eight intensities of flash stimulation were used (each intensity was repeated five times with an interval of at least 30 seconds; as the intensity increased, the interval was increased accordingly to ensure the recovery of photoreceptor cell function). The amplitude and latency of wave a (photoreceptor cell hyperpolarization) and wave b (bipolar cell depolarization) were recorded.

[0097] Photopic ERG: After completing the dark vision recording, turn on the background light (30 cd / m²). 2 Perform light adaptation for 5 minutes to allow the retina to transition to a state of near vision; then apply light at concentrations of 0.3, 1, 3, 10, and 30 cd·s / m². 2 Five intensities of flash stimulation were applied (each intensity was repeated three times with an interval of at least 10 seconds, and the interval time was increased with each increase in intensity), and the amplitude and latency of the photopic b wave (mediated by cone cells) were recorded.

[0098] Data Acquisition: Waveforms were automatically recorded three times for each stimulus intensity, and the average value was used as valid data. Mouse heart rate (≥300 beats / min) and respiration (≥60 breaths / min) were monitored simultaneously to ensure stable vital signs. ERG data were collected to compare Ubap1l under different stimulus intensities during dark and light adaptation. - / - Group and Ubap1l + / + Differences between groups were statistically analyzed using GraphPadPrism software, employing a two-tailed unpaired Student's t-test.

[0099] 3.3.2 Fundus photography experiment

[0100] Animal selection was the same as in 3.3.1 ERG experiment, i.e., Ubap1l animals aged 3, 6, and 9 months. - / - With Ubap1l + / + Mice, ≥6 mice per group.

[0101] Anesthesia and mydriasis procedures are the same as in the 3.3.1 ERG test (1% amobarbital intraperitoneal injection, compound tropicamide eye drops for mydriasis), ensuring that the pupil diameter is ≥2 mm.

[0102] Fundus photography: A Retinal Imaging Microscope (Micron IV, Phoenix, USA) was used, along with a mouse fixation table (37℃ insulated) and corneal contact lenses (to reduce aberrations). The mouse was fixed prone on the imaging table, and its head position was adjusted so that the center of the eyeball was aligned with the optical axis of the microscope. Hydroxypropyl methylcellulose eye drops were applied to the corneal surface to cover the cornea and prevent it from drying out. The focus was adjusted until the optic disc (a round, bright white structure with visible radial blood vessels) was clearly focused. Using the optic disc as the central reference point, scanning was performed in nine anatomical quadrants: optic disc center, superior (2 mm above the optic disc), inferior (2 mm below the optic disc), nose (2 mm nasal side of the optic disc), and temporal (2 mm temporal side of the optic disc); diagonally: superior nose (45°), superior temporal (135°), inferior nose (225°), and inferior temporal (315°).

[0103] 3.3.3 Retinal vibration sectioning and immunofluorescence experiment

[0104] Gene knockout mice (Ubap1l) ​​aged 3 months, 6 months, and 9 months were selected. - / - ) and wild-type mice (Ubap1l) + / + (n=3 per group) After perfusion with pre-chilled PBS (pH 7.4), the mice's eyeballs were quickly enucleated and placed in pre-chilled PBS culture dishes. Under a stereomicroscope, the outer layer of the eyeball was carefully separated using microforceps and ophthalmic scissors to expose the eyeball wall. The eyeball wall was cut along the limbus (corneoscleral junction), and the lens and vitreous body were removed, preserving the retinal pigment epithelium (RPE) / choroid complex. The neuroretina was gently dissected from the RPE / choroid layer using microforceps, and the separated neuroretina was temporarily stored in pre-chilled PBS. The separated retina was transferred to a mixture of 4% PFA and electron microscopy fixative (volume ratio 1:1) and immersed for 2 hours at room temperature. After fixation, the mice were washed three times with PBS (5 minutes each time with gentle agitation) to remove residual fixative. A 4% agarose gel (cooled to 40-50°C) was taken, and the retina was laid flat in the gel (ganglionic cell layer facing up), and slowly cooled until the gel was completely solidified. Fix the solidified agarose-retinal block onto the sample stage of the vibratory microtome, adjust the section orientation, and set the microtome parameters as follows: section thickness 100 μm, vibration frequency 30-50 Hz, and advance speed 0.1-0.3 mm / s (specific parameters need to be optimized according to the equipment model). Start the microtome, slowly advance the sample, collect the complete retinal section, and temporarily store it in a PBS culture dish (4℃, ≤24 hours).

[0105] Transfer sections to 24-well plates, add 0.5% Triton X-100 + 5% BSA mixture, and incubate on a shaker at room temperature for 1 hour to promote cell membrane permeability and block non-specific binding sites. Aspirate the blocking solution and wash three times with PBS; add diluted rhodopsin primary antibody and incubate overnight on a shaker at 4°C. After overnight incubation with primary antibody, wash three times with PBS; add fluorescent secondary antibody (species matched to the primary antibody), and incubate on a shaker at room temperature in the dark for 2 hours; aspirate the secondary antibody, wash three times with PBS, add DAPI working solution, and incubate at room temperature in the dark for 10 minutes to label cell nuclei. After DAPI staining, wash three times with PBS; transfer sections to glass slides using microtweezers, add anti-quenching mounting medium, cover with a coverslip (avoiding air bubbles), and store at 4°C in the dark (for imaging within ≤7 days).

[0106] Place the mounted slides on the stage of a Zeiss LSM 980 confocal microscope and adjust the focus until all layers of the retina are clearly visible. Set the excitation and emission channels (corresponding to the rhodopsin fluorescence secondary antibody channel, and DAPI corresponding to 405nm excitation), and the scanning parameters (resolution 1024×1024, Z-axis layer thickness 2-5μm). Perform multi-field acquisition of target areas (such as the outer segment of photoreceptors and rod cell-rich areas), and save them as TIFF or LSM format images for subsequent quantitative analysis (such as fluorescence intensity and cell count).

[0107] 4. Experimental Results

[0108] 4.1 Construction and Genotyping Results of Ubap1l Gene Knockout Mouse Model

[0109] Genotyping results showed that the genotype distribution of the F2 generation mice conformed to Mendelian inheritance laws, indicating that allele knockout had no germline lethal effect. Agarose gel electrophoresis confirmed that the gene knockout mice (Ubap1l)... - / - Only one knockout primer amplified a 378bp positive band, while the wild-type (Ubap1l) ​​band was different. + / + The mice showed only one 315bp positive band amplified by the wild-type primers, while the heterozygotes (Ubap1l) + / - The mouse showed two positive bands at 378 bp and 315 bp. Figure 4 As shown, Sanger sequencing results further confirmed the accuracy of the gene editing. Sequencing results from wild-type mice clearly revealed a continuous 315 bp sequence between exons 3 and 6; while homozygous samples showed a junctional sequence between introns 2 and 6, indicating that the target 8323 bp fragment was completely deleted with correct sequence ligation, and no non-specific insertions or deletions occurred. This result confirms at the molecular level that the Ubap1l gene function was successfully knocked out, and that the model construction was accurate and reliable.

[0110] 4.2 mRNA expression silencing validation results

[0111] like Figure 5 As shown, the real-time quantitative PCR results indicate that, compared with the wild type (Ubap1l) + / + Compared to mice, homozygous knockout (Ubap1l) - / - The relative expression level of Ubap1l mRNA in mouse retinal tissue was significantly reduced, almost to zero, indicating that the gene knockout model successfully achieved gene silencing at the transcriptional level.

[0112] 4.3 Electroretinography (ERG) Results

[0113] In electroretinography (ERG) testing, scotopic b-waves reflect the function of the rod cell system, while photopic b-waves represent the function of the cone cell system. Results showed that the ERG curves of wild-type mice at 3, 6, and 9 months of age all exhibited a normal pattern. Figure 6 E in Figure 7 E in Figure 8 E in the middle), and wild type (Ubap1l) + / + Compared to mice, Ubap1l - / - Mice showed progressive photocytic cell dysfunction from 3 months of age. For example... Figure 6 G in the image shows 3-month-old Ubap1l - / - In mice under dark adaptation, all stimulus intensities (0.003-10 cd·s / m) 2 The ERG amplitudes of all of them were significantly reduced (p<0.05), among which, such as Figure 6 The F in the figure shows 3-month-old Ubap1l - / - The peak amplitude of the maximum b-wave in mice was only 121.8 ± 21.8 μV, while that in wild-type mice reached 349.7 ± 133.0 μV. Further analysis showed that the progressive decrease in ERG scotoma b-wave amplitude followed a stimulus intensity-dependent pattern, reaching a peak amplitude at 0.003 cd·s / m². 2 The amplitude decreased by 2.96 times under stimulation, 10 cd·s / m 2 The stimulus decreased by a factor of 3.11 (p<0.05, linear mixture model). For example... Figure 7 G and Figure 8 As shown in G, Ubap1l at 6 months and 9 months of age - / - The mice exhibited more severe impairment in scotopic amplitude, with statistically significant differences in b-wave peak values ​​across all stimulus intensities. Even under high-intensity stimulation, the maximum b-wave peak value did not exceed 100 μV. Figure 7 F in Figure 8 In the photosensitive response (F). Figure 6 H in Figure 7 H in Figure 8 H in Ubap1l - / - The light-adapted b-wave in mice was 0.3 cd·s / m 2 The initial intensity begins to decrease (1.2 cd·s / m). 2 (Except for stimulation intensity), a decrease in amplitude was observed at all stimulation intensities; at 6 months and 9 months of age, the amplitude of photopic b-waves was still significantly lower than that of wild-type mice, and even at the highest stimulation intensity, it did not exceed 28.3±4.2 μV.

[0114] 4.4 Results of fundus photography

[0115] Fundus photography results showed that wild-type mice (Ubap1l) + / + Both eyes (OD right eye, OS left eye) showed normal retinal morphology at all ages, with even distribution of fundus blood vessels, normal color, and no abnormal patches. Figure 6 A and B in Figure 7 A and B in Figure 8 (A and B in the text).

[0116] Compared to wild-type mice, Ubap1l - / - Mice showed obvious pathological changes from 3 months of age, with significant pigmentary disturbances in the fundus of both eyes, accompanied by diffuse white spot degeneration and narrowing of blood vessels. Figure 6 (C and D in the original text). As the baby grows to 6 months, these lesions become more pronounced, the degenerated areas expand and merge, and the vascular network begins to thin. Figure 7 (C and D in the text). By 9 months of age, Ubap1l - / - The mice developed severe retinal lesions, with normal fundus structures almost entirely replaced by diffuse pigmentation and white lesions, exhibiting typical characteristics of late-stage retinal degeneration. Figure 8 (C and D in the text). This bilaterally symmetrical and progressively worsening pattern of the disease demonstrates that the deletion of the Ubap1l gene is the root cause of retinal degenerative changes.

[0117] 4.5 Results of observation of photoreceptor cell structure

[0118] like Figure 9 As shown in AB, CD, and EF, wild-type mice (Ubap1l) + / +The rod photoreceptors (green fluorescence) of wild-type mice exhibited a highly ordered morphological structure at all time points (3, 6, and 9 months of age). They were neatly and densely arranged, forming regular, uniformly long rod-shaped structures that clearly and parallelly connected with the nuclei of the inner nuclear layer (blue fluorescence) cells. Comparisons between different age groups showed that the retinal structure of wild-type mice remained stable with age, without obvious signs of degeneration, demonstrating the normal development and aging process of the retina in this strain of mice.

[0119] However, in gene knockout mice (Ubap1l) - / - In ), progressive and irreversible structural degradation was observed. At 3 months of age ( Figure 9 In GH (growth factor), compared with wild-type mice of the same age, knockout mice showed early pathological changes in rod photoreceptors, generally manifested as slight misalignment and slight unevenness in length; by 6 months of age ( Figure 9 In the IJ stage, the lesion severity significantly worsened, the overall structure of the rod photoreceptor became looser, the outer tip showed significant distortion, and the difference in rod length became more pronounced; by 9 months of age ( Figure 9 The KL (Kinshasa) in the rod has reached its most severe stage of degeneration. The structure of the outer segment of the photoreceptor is severely damaged, making it difficult to identify the complete cell morphology, and obvious twisting and deformation are visible in the outer segment.

[0120] In summary, this invention successfully constructed a stable, heritable Ubap1l gene knockout mouse model that accurately simulates the pathological process of human retinitis pigmentosa (RP). By precisely knocking out the splice sites from exon 3 to exon 6 and the coding region of the UBA2 domain using CRISPR-Cas9 technology, a complete loss of Ubap1l gene function was achieved. The effectiveness of the model was systematically verified at the molecular, functional, and phenotypic levels. This model not only successfully reproduced the core characteristics of RP, including progressive photocell dysfunction (significantly decreased ERG amplitude) and retinal structural lesions (fundus pigmentary disorders, white spot degeneration, and vascular attenuation), but also revealed the importance of the synergistic effect of the UMA-SOUBA linker and the SOUBA domain in maintaining protein function. This study fills the gap in Ubap1l gene defect disease models, providing a reliable and efficient in vivo research platform for in-depth research on the molecular mechanisms of RP, screening targeted drugs, and developing gene therapy strategies, and has significant scientific value and translational prospects.

[0121] The present invention has been further described above with reference to specific embodiments. However, it should be understood that the specific description herein should not be construed as limiting the nature and scope of the present invention. Various modifications made to the above embodiments by those skilled in the art after reading this specification are all within the scope of protection of the present invention.

Claims

1. A method of constructing a gene knockout mouse model, comprising the steps of: Ubap1l a) obtaining a mouse model of a disease; comprising the following steps: According to Ubap1l gene design sgRNA; the sgRNA includes sgRNA1 as shown in SEQ ID NO: 5 and sgRNA2 with sequence as shown in SEQ ID NO: 6; mixing sgRNA and Cas9 protein to obtain a gene editing solution; transferring the gene editing solution into mouse zygotes, and transplanting the zygotes transfected with the gene editing solution into pseudopregnant mice to obtain F0 generation mice; Screening based on F0 mice Ubap1l homozygous offspring to obtain Ubap1l homozygous mice with a gene knockout.

2. The construction method of claim 1, wherein, The knockout region targeted by the sgRNA covers Ubap1l the splice site of exon 3 to exon 6 of the gene and the UBA2 domain coding region.

3. The construction method of claim 1, wherein, The sgRNA1 and sgRNA2 are mixed at a molar ratio of 1:1-2.

4. The construction method of claim 3, wherein, Cas9 protein in the gene editing solution and sgRNA for targeting mouse Ubap1l sgRNA of the gene is mixed at a molar ratio of 1-2:

1.

5. The construction method of claim 2, wherein, The knockout region targeted by the sgRNA includes a SOUBA domain and a neighboring UMA-SOUBA connecting region.

6. The construction method of claim 1 wherein, The manner of transferring the gene editing solution into mouse zygotes is microinjection.

7. The construction method of claim 1 wherein, Screening based on F0 mice Ubap1l Methods of homozygous offspring of a gene include: backcrossing the F0 generation mice with germline transmission positive with wild type mice to obtain F1 generation heterozygous mice; The F1 hybrid mice obtained are intercrossed to obtain F2 homozygous mice, that is, the mice of the present application Ubap1l Gene knockout mouse model.

8. A construct constructed by the method of any one of claims 1-7 Ubap1l Use of a knockout mouse model for screening drugs for treating retinitis pigmentosa.

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