Application of IL10 gene editing in inflammatory bowel disease pig model

Abstract

The invention discloses an application of IL10 gene editing in an inflammatory bowel disease pig model. The gene editing is selected from gene knockout, gene knockdown, gene interference or gene point mutation capable of inactivating or reducing IL10 gene expression. It is proved for the first time that the IL10 <- / -> pigs obtained through gene editing can spontaneously suffer from enteritis in a conventional feeding environment, and a research tool closer to human physiological characteristics is provided; the IL10 < + / -> pig has no obvious inflammatory bowel disease phenotype in a conventional environment, the cost of model cultivation and propagation can be effectively reduced, a large animal model of the inflammatory bowel disease can be obtained through conditions induction and the like when needed, and different research requirements can be flexibly met; the IL10 < + / -> pig can be used as a model for researching IBD pathogenesis inducements and related mechanisms of people with low IL10 expression.

Description

Technical Field

[0001] This invention relates to the field of animal genetic engineering technology, specifically to the application of gene editing in inflammatory bowel disease models, particularly the application of IL10 gene editing in inflammatory bowel disease pig models. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic inflammatory bowel disease with complex and diverse etiologies. The pathogenesis of IBD is complex and not fully understood, and treatment options are very limited. In order to further elucidate the pathological mechanisms, screen highly effective targeted drugs, and optimize treatment regimens, relevant disease evaluation and research models are urgently needed.

[0003] For many years, IL10 knockout mice have been a classic IBD disease model (see Kühn et al. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75, 263-274 (1993)). They exhibit typical IBD symptoms such as shortened colon and impaired intestinal barrier, making them an important tool for elucidating the etiology of IBD and developing drugs. However, existing IL10 knockout mice have significant limitations: mice differ significantly from humans in intestinal anatomy, immune composition, gut microbiota, and metabolic characteristics, severely limiting the clinical translational value of related drug research. There is an urgent need for larger animal models that are more closely similar to humans.

[0004] Chinese patent application CN114941014A discloses a method for constructing a hamster model based on IL-10 gene knockout, including designing a hamster IL-10 gene-specific targeting sequence, preparing the cas9 mRNA and sgRNA; then collecting and culturing hamster zygotes; next, co-injecting the sgRNA and cas9 mRNA into the cytoplasm of the hamster zygotes; finally, implanting the microinjected zygotes into surrogate hamsters, resulting in the birth of F0 generation hamsters. This invention achieves this by knocking out the IL-10 gene in hamsters (IL-10...). - / - The study of IL-10 and genetic modification may provide strong experimental evidence for the mechanism by which IL-10 affects lipid metabolism and atherosclerosis (AS), clarify the pathophysiological significance and clinical value of IL-10 in AS and cardiovascular diseases, and provide new academic perspectives, drug targets and intervention strategies for the prevention and treatment of cardiovascular diseases.

[0005] From this document Figure 1 According to the disclosed knockout strategy, the technology knocks out 9 bp of exon 2 of the IL10 gene; the technology in this paper is used to construct models of cardiovascular diseases such as atherosclerosis (AS).

[0006] Chinese patent application CN112553254A discloses an IL10 gene knockout mouse model and its construction method and application. The construction method includes the following steps: S1, based on CRISPR / Cas9 technology, the knockout region of the IL10 gene is determined, and specific target sites gRNA1 and gRNA2 are designed according to the determined knockout region. The gene sequences of gRNA1 and gRNA2 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; S2, active gRNA1, gRNA2, and Cas9 proteins are microinjected into mouse zygotes, and the surviving zygotes are transplanted into pseudopregnant female mice. After the female mice become pregnant and give birth, F0 generation mice are obtained; S3, the F0 generation mice obtained in step S2 are mated with wild-type mice to obtain F1 generation heterozygotes; S4, the F1 generation heterozygotes obtained in step S3 are inbred to obtain F2 generation mice, which are the IL10 gene knockout mouse model.

[0007] As described in the examples in this literature, knocking out all exons 2-5 of the mouse IL10 gene resulted in F1 generation heterozygous mice with deletions of 4888 bp or 4885 bp. Although the literature claims that the gene-edited mouse model can be used in studies related to colitis, tumors, immunity, and inflammation, it did not specifically test the relevant model indicators or the results of its application in specific studies, and the specific effects of this gene-edited mouse model remain unclear.

[0008] Furthermore, studies have shown that IL10 gene knockout in chickens does not lead to IBD (see Meunier, D., et al. (2025) Interleukin 10 controls the balance between tolerance, pathogen elimination, and immunopathology in birds. eLife, 14, RP106252.). This indicates that the IL10 gene knockout-induced enteritis phenotype is species-specific, and different species may exhibit different phenotypes after IL10 gene knockout, which poses a challenge to the construction of large animal models of IBD.

[0009] Pigs are highly similar to humans in terms of intestinal anatomy, immunological characteristics, gut microbiota composition, and clinical assessment methods, making them one of the preferred species for constructing large animal models. However, it remains unclear whether knocking out the pig IL10 gene can induce the IBD phenotype and how to knock it out (e.g., whether to knock out completely or partially, and which parts to knock out, etc.), and there are no reports of related pig animal models. Summary of the Invention

[0010] The purpose of this invention is to provide the application of porcine IL10 gene editing in an inflammatory bowel disease model, which is constructed by editing the porcine IL10 gene, and to confirm that IL10 gene knockout homozygotes (IL10) - / - The spontaneous development of inflammatory bowel disease under normal conditions provides a good animal model for the research, development, and evaluation of therapeutic drugs for IBD. In addition, a heterozygous IL10 gene knockout pig (IL10) was also constructed. + / - Under normal conditions, they do not exhibit obvious characteristics of inflammatory bowel disease, and homozygous IL10 gene knockout pigs can be obtained through breeding.

[0011] The technical solution adopted in this invention is as follows:

[0012] In a first aspect, the present invention provides the application of IL10 gene editing in a pig model of inflammatory bowel disease, characterized in that the gene editing is selected from gene knockout, gene knockdown, gene interference or gene point mutation that can inactivate or reduce the expression of the IL10 gene.

[0013] Preferably, the gene editing employs the CRISPR / Cas9 gene editing system, which specifically includes the following steps:

[0014] (1) Extract porcine genomic DNA, design specific amplification primers based on the porcine IL10 gene, and amplify the IL10 gene. The nucleotide sequence of the IL10 gene is as shown in SEQ ID NO:1 or has an identity of not less than 90% with it.

[0015] (2) Design a specific sgRNA guide sequence targeting the porcine IL10 gene based on the porcine IL10 gene, wherein the sgRNA guide sequence includes any combination of two of the nucleotide sequences shown in SEQ ID NO.10-23;

[0016] (3) The RNP complex formed by the sgRNA guide sequence and Cas9 protein described in step (2) was transfected into porcine fetal fibroblasts by electroporation.

[0017] (4) IL10 gene-edited porcine fetal fibroblasts were obtained by flow cytometry sorting and gene identification.

[0018] Preferably, the specific amplification primers in step (1) are one of the primer pairs consisting of any two primers shown in the following sequence numbers: SEQ ID NO:2 and SEQ ID NO:3; SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7; SEQ ID NO:8 and SEQ ID NO:9.

[0019] More preferably, the sgRNA guide sequence in step (2) is a combination of the nucleotide sequences shown in SEQ ID NO:13 and SEQ ID NO:19.

[0020] Preferably, the gene editing further includes the following steps:

[0021] (5) Preparation of reconstructed embryos: Using the IL10 gene-edited porcine fetal fibroblasts obtained in step (4) as donor cells, oocytes were collected and enucleated. Then, the donor cells and oocytes were recombined using somatic cell nuclear transfer technology to obtain reconstructed embryos.

[0022] (6) Reconstructed embryo fusion activation: The reconstructed embryo is activated to obtain a reconstructed IL10 gene-edited pig embryo that can develop;

[0023] (7) Embryo transfer: The activated reconstructed embryos are transferred into the uterus of pigs to obtain IL10 gene-edited pigs.

[0024] Preferably, the method for identifying the obtained IL10 gene-edited pig is as follows: extract genomic DNA from its ear margin tissue, and then identify the IL10 gene target fragment by PCR amplification using the specific amplification primers.

[0025] Preferably, in the identification step, if the IL10 gene electrophoresis band obtained by PCR amplification has only one band corresponding to the size of the gene knockout band, the identification result is IL10- / - gene knockout homozygote; if the electrophoresis band has two bands, one corresponding to the size of the gene knockout band and the other corresponding to the size of the wild-type band, the identification result is IL10- / - gene knockout heterozygote. + / - .

[0026] Secondly, the present invention provides sgRNA guide sequence combinations for the above-described applications of the present invention, characterized in that the sgRNA guide sequence comprises any two of the nucleotide sequences shown in SEQ ID NO.10-23.

[0027] Preferably, the sgRNA guide sequence is a combination of the nucleotide sequences shown in SEQ ID NO:13 and SEQ ID NO:19.

[0028] The present invention also provides a biomaterial, characterized in that the biomaterial contains: 1) the sgRNA guide sequence combination of the present invention, and / or 2) one or more of a vector, expression cassette or gene-editing cell comprising the sgRNA guide sequence combination of the present invention.

[0029] The present invention has the following beneficial effects:

[0030] This invention is the first to demonstrate that knocking out the IL10 gene in pigs can effectively construct an IBD model. Compared with the classic IBD model (i.e., the IL10 gene knockout mouse model), this model has a more human-like phenotype, which provides a more reliable animal model for overcoming the bottlenecks in IBD pathogenesis research and drug development.

[0031] This invention is the first to demonstrate the ability to obtain homozygous pigs (IL10) by knocking out a partial sequence of the IL10 gene in pigs. - / - Inflammatory bowel disease can spontaneously occur under normal feeding conditions, providing a large animal model that is closer to human physiological characteristics for inflammatory bowel disease research.

[0032] Simultaneously, this invention also marks the first discovery of IL10 gene knockout heterozygous pigs (IL10... + / - The expression level of IL10 protein in the pig was significantly reduced, but no obvious inflammatory bowel disease phenotype was observed under normal conditions. This heterozygous pig can obtain IL10 through conventional breeding. ⁻ / ⁻ Pigs thus directly obtained spontaneous IBD models, which can also be used as models to study the pathogenesis and related mechanisms of IBD in people with low IL10 expression, further expanding the application scenarios of this gene-edited pig model.

[0033] Brief description of the illustrations in the instruction manual

[0034] Figure 1 This invention demonstrates a partial screening method for dual sgRNA pairing that specifically targets porcine IL10.

[0035] Figure 2 The results of clonal PCR identification of IL10 gene-edited porcine fetal fibroblasts of this invention are shown.

[0036] Figure 3 The sequence alignment diagram of the IL10 gene-edited porcine fetal fibroblast monoclonal cells of this invention is shown.

[0037] Figure 4 The image shows piglets produced by somatic cell nuclear transplantation of IL10 gene-edited porcine fetal fibroblasts according to the present invention.

[0038] Figure 5 The results of gene PCR identification of the IL10 gene-edited pigs of this invention are shown.

[0039] Figure 6 The results of the Sanger sequencing alignment analysis of the IL10 gene-edited pigs of this invention are shown.

[0040] Figure 7 This invention demonstrates the use of GAPDH as an internal control in RT-PCR to analyze IL10 gene knockout homozygous pigs (IL10). - / -mRNA expression status. Figure A shows the PCR results identified using IL10 gene-specific mRNA primers, and Figure B shows the PCR results identified using GAPDH primers as a GAPDH internal control.

[0041] Figure 8 This invention demonstrates the IL10 gene knockout heterozygous pig (IL10). + / - The IL10 secretion level of PBMCs after in vitro stimulation with TLR2 ligand (Pam3CSK4). In the figure, group 1 represents the cell supernatant detection results after 24 hours of culture without Pam3CSK4, group 2 represents the cell supernatant detection results after 24 hours of culture with Pam3CSK4, group 3 represents the cell supernatant detection results after 48 hours of culture without Pam3CSK4, group 4 represents the cell supernatant detection results after 48 hours of culture with Pam3CSK4, and group 5 represents the plasma detection results.

[0042] Figure 9 The results show representative blood routine examinations of the IL10 gene-edited pigs of this invention.

[0043] Figure 10 This invention demonstrates the IL10 knockout homozygous pigs (IL10) - / - The results of colon length analysis are shown in Figure A (photographs) and Figure B (length data analysis).

[0044] Figure 11 This invention demonstrates the IL10 knockout homozygous pigs (IL10) - / - ) Colonic pathological analysis results. Among them, Figures A and D are HE stained 100x microscopic images, Figures B and E are HE stained 400x microscopic images, Figures C and F are PAS stained 400x microscopic images. Green arrows indicate mucosal epithelial cell shedding and necrosis, red arrows indicate congestion, yellow arrows indicate plasma cell infiltration, black arrows indicate lymphocyte infiltration, and blue arrows indicate goblet cells.

[0045] Figure 12 This invention demonstrates the IL10 knockout pigs (IL10) of the present invention. - / - pig Enteritis model, classic IBD animal model (Il10 knockout mouse (Il10)) - / - Differential gene similarity analysis at the transcriptome level was performed on a mouse enteritis model and human IBD patients (UC type, human UC). Figures A, B, and C show the differences between human UC and IL10, respectively. - / - Venn diagrams of differentially expressed genes, upregulated differentially expressed genes, and downregulated differentially expressed genes in pigs; Figures D, E, and F show the differences between human UC and Il10, respectively. - / - Venn diagrams of differentially expressed genes, upregulated differentially expressed genes, and downregulated differentially expressed genes in mice.

[0046] Figure 13 This invention demonstrates the IL10 knockout pigs (IL10) of the present invention. - / - Swine enteritis model, classic IBD animal model (IL10 knockout mice (IL10) - / - KEGG enrichment pathway analysis of differentially expressed genes at the transcriptome level in a mouse enteritis model and human IBD patients (UC type, human UC) was performed. The raw graphs were obtained from the data output of Metware Sequencing (https: / / cloud.metware.cn). Figures A and B show the correlation between human UC and IL10, respectively. - / - Bubble charts of KEGG enrichment analyses for upregulated and downregulated differentially expressed genes in pigs; Figures C and D show the KEGG enrichment analyses for human UC and Il10, respectively. - / - Bubble chart showing KEGG enrichment analysis of upregulated and downregulated differentially expressed genes in mice. The vertical axis represents the KEGG pathway; the horizontal axis represents the enrichment factor, which is the ratio of the number of differentially expressed genes annotated under that entry to the total number of genes annotated under that entry. The larger the enrichment factor, the greater the enrichment. The size of the dot represents the number of genes enriched in that pathway; the more genes, the larger the dot. The color of the dot represents the significance of the enrichment (Q-value): the closer the Q-value is to 0, the more reliable the result; the red bubbles in the chart have Q-values ​​close to 0, indicating the highest enrichment significance. Detailed Implementation

[0047] The following detailed description provides further details through specific embodiments. However, it should be noted that the embodiments described below are merely for illustrating the content of the invention and do not represent that the invention is limited to the described embodiments. Therefore, non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the above-described invention still fall within the protection scope of the invention, and the scope of protection of the appended claims shall prevail.

[0048] Those skilled in the art will understand that the IL10 cytokine is encoded by the IL10 gene. In this invention, gene editing is performed using the porcine IL10 gene as the target gene. The full-length sequence of this gene is known to those skilled in the art. It is also understood that IL10 generally refers to the protein it encodes, interleukin-10.

[0049] Those skilled in the art will understand that the gene editing described in this invention refers to any gene editing method that can inactivate or reduce the expression of the IL10 gene, such as gene knockout, gene knockdown, gene interference, or gene point mutation. These gene editing methods can be achieved through traditional means such as PCR tools or kits, or through specialized gene editing platforms (such as CRISPR / Cas, ZFN, TALEN, etc.). These technologies are well known to those skilled in the art.

[0050] Although this article only uses the relevant genes of Bama pigs as an example to verify the effectiveness of the related technologies of the present invention through the CRISPR / Cas gene editing system, those skilled in the art will understand that most sequences of the same genes in animals are conserved, especially in pigs. The IL10 gene target sequence involved in this invention is highly conserved in various pig breeds. Therefore, the technology of the present invention is also applicable to other pig breeds. Other gene editing methods are also known to achieve the same effect of inactivating or reducing IL10 gene expression. Therefore, the technology of the present invention is also applicable to other gene editing methods.

[0051] Those skilled in the art will understand that the CRISPR / Cas system is a next-generation gene editing technology with extremely wide and mature applications. In the embodiments of this invention, the type II CRISPR / Cas system, namely CRISPR / Cas9, is used, which is currently the most widely used CRISPR / Cas system. However, those skilled in the art will also understand that other mature CRISPR / Cas gene editing systems or other gene editing systems can be used. These editing systems are all commercially available or licensed, and their operating methods are mature, not affecting the implementation of the technical solution of this invention.

[0052] Those skilled in the art will understand that the specific applications of the animal models of the present invention include, but are not limited to, basic research on the pathogenesis of porcine inflammatory bowel disease, drug screening and research for the prevention or treatment of porcine inflammatory bowel disease, and related medical teaching or training.

[0053] Unless otherwise specified in this invention, any other technologies, instruments, equipment, and materials known to those skilled in the art that can achieve the same purpose may be used. Even if the technologies, instruments, equipment, and materials used in this invention are specifically specified, it does not mean that this invention can only use these technologies, instruments, equipment, and materials, but merely represents the preferred solution of this invention. Those skilled in the art can still use any other technologies, instruments, equipment, and materials known to those skilled in the art that can achieve the same purpose.

[0054] The following are the sequence descriptions corresponding to the sequence numbers in the sequence list:

[0055] The nucleotide sequence of the IL10 gene (gene accession number: 397106) in the NCBI database is shown in SEQ ID NO:1.

[0056] Specific primers for amplifying or identifying the porcine IL10 gene are provided. These primer pairs can specifically amplify the target DNA fragment. The sequence descriptions, numbers, and specific sequences of these primers, as well as the theoretical values ​​for the band sizes obtained from PCR amplification, are shown in the table below:

[0057] Table 1: Primer sequences for IL10 target region-specific amplification

[0058]

[0059] sgRNA guide sequences are one of the key factors affecting the effectiveness of CRISPR / Cas gene editing systems. This invention names 14 sgRNA guide sequences specifically targeting the porcine IL10 gene as IL10-sgRNA1-14 (Table 2). In the sequences provided by this invention, the base sequence orientation is all from 5' to 3'.

[0060] Table 2: IL10 sgRNA guide sequence

[0061]

[0062] Example 1: Construction of IL10 gene-edited pigs

[0063] 1. Construction and identification of IL10 gene-edited porcine fetal fibroblast cell lines

[0064] (1) Design and acquisition of sgRNA sequences:

[0065] Genomic DNA was extracted from Bama pigs, and primers were designed to amplify the target sequence of the IL10 gene (4 pairs of primers, see Table 1, all of which amplified the target sequence of the IL10 gene). Sequencing of the target sequence was performed using primer IL10-04, followed by alignment analysis. The results showed that the Bama pig IL10 gene sequencing fragment (4329 bp) obtained in this invention had 17 base differences and 4 gaps (3 1 bp gaps, 1 10 bp gap, total gap length 13 bp) compared with the 558-4893 region of the porcine IL10 gene (gene ID: 397106) via BLAST sequence alignment, with a sequence identity of 99.3%. sgRNAs were designed using CRISPOR (http: / / crispor.tefor.net / ), and 14 sgRNA guide sequences targeting the IL10 gene were designed and synthesized by Nanjing Genscript Biotech Co., Ltd.

[0066] (2) Construction of IL10 gene-edited porcine fetal fibroblast cell line:

[0067] Using Knockout TM Resuscitate porcine fetal fibroblasts (P1 generation, isolated and preserved by Chongqing Academy of Animal Sciences) in DMEM complete medium (Gibco). After the cell density reaches 70-90%, passage them. After 2-4 days of culture, when the cell density reaches 80-90%, use them for transfection.

[0068] 1) sgRNA pairing and screening:

[0069] Transfection was performed using electroporation.

[0070] i. Divide 1×10 6 One cell was used for electroporation to test paired sgRNA. The cells were washed twice with DPBS (Dulbecco's Phosphate-Buffered Saline) and resuspended in 87 μL opti-MEM medium (Gibco).

[0071] ii. Preparation of RNP complex: Mix 5 μg of each of the paired IL10 gene double sgRNAs with 6 μg of Cas9 protein (M0646, NEB) and incubate at room temperature for 15 min to form RNP complex (see Table 3 below).

[0072] Table 3: Composition of the RNP complex for IL10 gene editing

[0073]

[0074] iii. Electroporation:

[0075] After mixing the RNP complex with porcine fetal fibroblasts, 100 μL was added to an electroporation cuvette and the NEPA21 high-efficiency gene transfection system (NEPA GENE Co., Ltd) was used with the following parameters set:

[0076] Poring Pulse: Voltage: 150 V; Pulse Duration: 5 ms; Pulse Interval: 5 ms; Number of Pulses: 2; Attenuation Rate: 10%; Polarity: +

[0077] Transfer Pulse: Voltage: 20 V; Pulse Duration: 50 ms; Pulse Interval: 50 ms; Number of Pulses: 5; Attenuation Rate: 40%; Polarity: + / -

[0078] Click Ω to record the impedance value (range 30-50Ω).

[0079] iv. Add the electrotransfected cells to 6-well plates and incubate for 24 hours;

[0080] v. PCR detection of targeting efficiency: Based on the designed sgRNA guide sequence position, the specific amplification primer sequences used to identify the IL10 gene targeting region are shown in Table 1.

[0081] vi. 24 hours after electroporation, genomic DNA was extracted from the cells, and PCR amplification targeting the IL10 gene was performed using the specific amplification primers described above as identification primers. The PCR reaction system (Takara) was 50 μL: 2×PrimeSTAR MaxPremix: 25 μL, F: 0.2 μM, R: 0.2 μM, DNA: 20-100 ng, H2O added to 50 μL. The PCR reaction conditions were: 98℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 62℃ annealing for 5 s, 72℃ extension for 5 s, for a total of 35 cycles, followed by a final extension at 72℃ for 5 min. The obtained PCR products were subjected to agarose gel electrophoresis. Analysis showed that the double sgRNA targeting the IL10 gene showed correct bands on electrophoresis, with the 20# combination showing a targeting efficiency of 23.83% (see...). Figure 1 ), and selected combination 20# for the next step of constructing IL10 gene-edited porcine fetal fibroblast cell line.

[0082] Based on the screening of the above sgRNA targeting conditions, the optimal sgRNA combination targeting the porcine IL10 gene (IL10-sgRNA4 + IL10-sgRNA10) was determined.

[0083] 2) Screening of IL10 gene-edited cell lines:

[0084] i. The optimal sgRNA combination (IL10-sgRNA4 + IL10-sgRNA10) selected above was electrotransfected into porcine fetal fibroblasts and then cultured in 6-well plates for 24 hours.

[0085] ii. Twenty-four hours after electroporation, genomic DNA was extracted from a portion of the cells, and the targeting efficiency of the IL10 gene was verified by PCR amplification using identification primers. The PCR reaction system (Takara) was 50 μL: 2×PrimeSTAR Max Premix: 25 μL, F: 0.2 μM, R: 0.2 μM, DNA: 20-100 ng, and H2O was added to 50 μL. The PCR reaction conditions were: 98℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 62℃ annealing for 5 s, 72℃ extension for 5 s, for a total of 35 cycles, followed by a 72℃ extension for 5 min.

[0086] iii. After digesting the cells that have passed the targeting efficiency test, use flow cytometry to separate individual cells into 96-well plates and culture for 10-13 days, changing the medium every 3-4 days.

[0087] iv. After passage of single clones with a confluence of 90% into 24-well plates, continue culturing for 2-3 days, and perform preservation and PCR identification on the clones in the 24-well plates.

[0088] v. Genomic DNA was extracted from a subset of cells, and the IL10 gene was identified by PCR using identification primers. The PCR reaction system (Takara) was 50 μL: 2×PrimeSTAR Max Premix: 25 μL, F: 0.2 μM, R: 0.2 μM, DNA: 20-100 ng, H2O added to 50 μL. The PCR reaction conditions were: 98℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 62℃ annealing for 5 s, 72℃ extension for 5 s, for a total of 35 cycles, followed by a final extension at 72℃ for 5 min. The results of partial clone PCR detection are shown below. Figure 2 As shown in the figure, PCR amplification of homozygous IL10 gene-edited cells produced one mutant band with a size of 2579 bp, while PCR amplification of heterozygous IL10 gene-edited cells produced two bands, with the mutant band at 2579 bp and the wild-type band at 4335 bp.

[0089] i. Select monoclonal cells and perform sequencing analysis on the edited PCR products. The sequencing results are shown below. Figure 3 The results showed that, compared with the control group, the IL10 gene fragment in the edited cells was deleted, with a deletion size of 1751 bp.

[0090] (3) Constructing IL10 gene-edited pigs:

[0091] Using the IL10 gene-edited pig fetal fibroblasts constructed above as donor cells, somatic cell nuclear transfer was performed to prepare IL10 gene-edited pigs: oocytes were collected and matured in vitro. The nucleus and first polar body of the oocytes were removed using a micromanipulator. Donor cells were selected and injected into the zona pellucida to complete embryo reconstruction and obtain cloned embryos. Electrofusion activation was performed, and the activated cloned embryos were transferred to embryo culture medium for culture. Well-developed cloned embryos were transferred into the pig uterus for pregnancy to obtain IL10 gene-edited pigs. Figure 4 ).

[0092] Example 2: Identification of IL10 gene knockout pigs

[0093] (1) Extraction of genomic DNA and PCR identification

[0094] Ear margin tissue was collected from IL10 gene-edited pigs, and genomic DNA was extracted from the ear margin tissue using a genomic DNA extraction kit (Qiagen). Using the extracted ear margin tissue genomic DNA as a template, PCR amplification was performed (specific amplification primers were selected from Table 1), and the products were analyzed by electrophoresis. The results showed ( Figure 5 ), 1-5# are all homozygous IL10 gene knockout pigs ( Figure 5 A), the band size was 2324 bp; 0101-0107 were all IL10 gene knockout pig heterozygotes ( Figure 5 (B) The two PCR bands were 4084 bp and 2324 bp, respectively.

[0095] (2) Sanger sequencing

[0096] The PCR products obtained above were subjected to Sanger sequencing, and the sequencing results were compared and analyzed. Figure 6 The results showed that IL10 gene knockout homozygous pigs (IL10) - / - A 1751 bp deletion occurred in exon 4-5 of the porcine IL10 gene, which is in line with expectations.

[0097] (3) IL10 gene knockout homozygous pigs (IL10 - / - Identification of IL10 mRNA levels in )

[0098] Collect IL10 gene knockout homozygous pigs that developed spontaneous inflammatory bowel disease (IL10). - / - Mesenteric lymph nodes were used to extract total RNA, which was reverse transcribed into cDNA. mRNA primer sequences were designed (see Table 4 below). RT-PCR was performed using GAPDH as an internal control to analyze the IL10 gene knockout homozygous pigs (IL10). - / - mRNA expression status, results as follows Figure 7 IL10 gene knockout homozygous pigs (IL10 - / - IL10 was not expressed at the mRNA level.

[0099] Table 4: Primers for mRNA identification

[0100]

[0101] (4) IL10 gene knockout heterozygous pigs (IL10 + / - Identification of IL10 protein levels in )

[0102] To detect the IL10 protein expression level in IL10 gene knockout heterozygous pigs, peripheral blood mononuclear cells (PBMCs) were first isolated and cultured in vitro. Then, they were stimulated with Toll-like Receptor 2 Ligand (TLR2 Ligand) Pam3CSK4, and the IL10 protein expression level was detected by ELISA analysis of the cell supernatant.

[0103] The specific steps are as follows:

[0104] 1) Isolation of porcine peripheral lymphocytes

[0105] 10 mL of peripheral blood was collected from IL10 gene-edited pigs and separated using porcine peripheral blood lymphocyte separation medium KIT (LTS1110, Tianjin Haoyang Biological Products Technology Co., Ltd.). The specific procedures are as follows:

[0106] a) Dilution: Dilute 1 part PBS with 2 parts blood (PBS: anticoagulated blood = 1:2); Note: Diluent requirements: Use a buffer or culture medium free of calcium and magnesium ions for blood dilution.

[0107] b) Take a sterile 50 mL centrifuge tube, add an equal volume of separation buffer (diluted blood volume: separation buffer volume = 1:1), and then slowly add the blood sample or fresh anticoagulated blood. The diluted blood sample should be slowly and carefully added to the separation buffer interface.

[0108] c) Centrifugation: Centrifuge at 650 g for 30 min at 20℃, with slow speed reduction enabled;

[0109] d) After centrifugation, the centrifuge tube separates into four layers from top to bottom. The first layer is the plasma layer. The second layer is a ring-shaped, milky-white layer of porcine lymphocytes. The third layer is the clear separation fluid layer. The fourth layer is the red blood cell layer.

[0110] e) Carefully aspirate the top 2 / 3 of the first layer of plasma and label it as pig plasma;

[0111] f) Carefully aspirate the second layer of lymphocytes into a new 50 mL centrifuge tube using a Pasteur pipette and add 10 mL of washing solution;

[0112] g) Centrifugation: Centrifuge at 250 g for 10 min at 20℃;

[0113] h) Discard the supernatant and resuspend the cells in 10 mL of washing buffer;

[0114] i) Centrifugation: Centrifuge at 250 g for 10 min at 20℃;

[0115] j) Discard the supernatant, add 3-5 times the volume of red blood cell lysis buffer, and incubate on ice for 3 min;

[0116] k) Centrifugation: Centrifuge at 250 g for 5 min at 20℃;

[0117] l) Discard the supernatant, add 10 mL of PBS containing 2% FBS to resuspend the cells, and wash twice;

[0118] m) Centrifugation: Centrifuge at 250 g for 5 min at 20℃;

[0119] n) Discard the supernatant, add 2 mL of PBS containing 2% FBS to resuspend the cells, and count them using a cell counting chamber;

[0120] o) The isolated peripheral lymphocytes were processed at a concentration of 1×10⁻⁶ 7 / The tubes are frozen;

[0121] 2) In vitro stimulation of PBMCs:

[0122] Recovery IL10 + / - PBMCs of pigs and common wild-type Bama pigs, at 1×10 6 Cells were seeded into 24-well plates and cultured at 37°C for 2 hours. TRL2 ligand (Pam3CSK4, 50 ng / mL, Invivogen) was added to the experimental group and cultured at 37°C. Cell supernatants were collected at 24 and 48 hours for ELISA detection.

[0123] 3) ELISA detection of IL10 protein expression level:

[0124] The supernatant collected in the previous step was analyzed using the Pig IL10 ELISA kit (Solarbio). The results are as follows: Figure 8 Image 1 shows the cell supernatant detection results after 24 hours of culture in the group without Pam3CSK4; image 2 shows the cell supernatant detection results after 24 hours of culture in the group with Pam3CSK4; image 3 shows the cell supernatant detection results after 48 hours of culture in the group without Pam3CSK4; image 4 shows the cell supernatant detection results after 48 hours of culture in the group with Pam3CSK4; and image 5 shows the plasma detection results. This demonstrates that the IL10 protein expression level is significantly reduced in IL10 gene knockout heterozygous pigs.

[0125] Example 3: Phenotypic Analysis of IL10 Gene-Edited Porcine Inflammatory Bowel Disease

[0126] Homozygous IL10 gene knockout pigs (IL10 - / - When placed in a standard environment, animals spontaneously develop inflammatory bowel disease, characterized by the following:

[0127] (1) Anemia and inflammatory response

[0128] IL10 samples collected from spontaneously occurring inflammatory bowel disease - / - A complete blood count (CBC) was performed on whole blood from pigs, and the results showed ( Figure 9 ), IL10 - / - The extremely low hemoglobin content in pig blood, resulting in anemia, along with an increased total white blood cell count and elevated platelet count, indicates an inflammatory response in the body.

[0129] (2) Shortened colon

[0130] IL10 samples collected from spontaneously occurring inflammatory bowel disease - / - Compared with the same-month-old control group, the pig colon ( Figure 10 It has become noticeably shorter.

[0131] (3) Severe diarrhea

[0132] IL10 samples collected from spontaneously occurring inflammatory bowel disease - / - Pig colons were stained with HE and PAS staining and the staining was visible ( Figure 11 The colonic mucosal epithelial cells showed obvious shedding and necrosis, congestion, and severe inflammatory cell infiltration. PAS staining showed a significant reduction in goblet cells.

[0133] (4) Similarity analysis of IL10-edited pig and human IBD at the transcriptome level

[0134] To investigate whether IL10 knockout pigs are more closely related to the pathogenesis of human IBD than the classic IBD model (i.e., IL10 knockout mice), this invention compares their similarity to human IBD. We collected IL10 knockout homozygous pigs (IL-10 knockout mice). - / -The colon during spontaneous enteritis was examined and transcriptomic analysis was performed. Human IBD can be divided into two core subtypes: ulcerative colitis (UC) and Crohn's disease (CD), with UC lesions primarily located in the colon. This invention cites research data from human IBD patients (UC type, human UC) GSE109142 and GSE117993 (see Haberman Y, Karns R, Dexheimer PJ, Schirmer M, et al. Ulcerative colitis mucosal transcriptomes reveal mitochondriopathy and personalized mechanisms underlying disease severity and treatment response. Nat Commun. 2019 Jan 3;10(1):38.), and also cites research data from IL10 knockout mice GSE264294 (see Li L, Ma C, Chen K, Jia Y, Wu Y, et al. Integrated transcriptomic and proteomic profiling of colonic tissue in interleukin-10-deficient mice. SciData. 2025 Jul 1;12(1):1109.). A comparison of pig and human data reveals that IL10... - / - There are 646 differentially expressed genes between pig and human IBD patients (UC type, human UC). Figure 12 A), of which a total of 333 differentially regulated genes were upregulated ( Figure 12 B), a total of 174 differentially regulated genes were downregulated. Figure 12 C); A comparison of mouse and human data revealed that IL10 - / - There are 231 differentially expressed genes between mouse and human IBD patients (UC type, human UC). Figure 12 D), of which a total of 144 differentially regulated genes were upregulated ( Figure 12 E), a total of 26 differentially regulated genes were downregulated ( Figure 12 F).

[0135] Further KEGG enrichment analysis of co-upregulated and downregulated differentially expressed genes revealed that IL10 - / -In both porcine and human IBD patients (UC type, human UC), the KEGG enrichment pathways with upregulated genes are concentrated in the PI3K-Akt signaling pathway, cytokine-cytokine receptor interaction, interleukin-17 signaling pathway, tumor necrosis factor signaling pathway, and mitogen-activated protein kinase signaling pathway. Figure 13 A) The enriched pathways include signal transduction, immune / inflammation-related pathways, cell adhesion / matrix interaction pathways, and disease-related pathways (cancer pathways, diabetic complication pathways), indicating that IL10 - / - The biological processes involved in differentially expressed genes in pigs are closely related to signal regulation, immune responses, cellular function, and disease development, and are highly consistent with the inflammatory dysregulation and immune imbalance in human IBD patients (UC type, human UC); IL10 - / - The KEGG enrichment pathways of downregulated genes in pigs are concentrated in metabolic pathways, mineral absorption, and tight junctions. Figure 13 B), the enriched pathways are mainly metabolic and absorption-related, while also involving cellular function (tight junctions, cell adhesion molecules), disease-related pathways (insulin resistance, juvenile diabetes), and the peroxisome proliferator-activated receptor signaling pathway (PPAR signaling pathway), indicating that IL10 - / - The functions of differentially expressed genes in pigs are concentrated in metabolism, nutrient absorption, and related physiological and pathological processes, closely resembling the core downregulation characteristic of "weakened nutrient absorption" in human IBD patients (UC type, human UC). Meanwhile, the shared upregulation pathways in IL10 gene knockout mice and humans are mainly related to neurodegenerative diseases and infection. Figure 13 C), with a weaker association with core inflammatory and immune pathways in human IBD patients (UC type, human UC); downregulated pathways focus on signaling and metabolic pathways ( Figure 13 D), but deviates from the core downregulated feature of "nutrient absorption" in human UC. The above analysis shows that the molecular pathway characteristics of IL10 gene knockout pigs are more consistent with the pathological mechanism of human IBD patients (UC type, human UC), making them a more ideal large animal model than IL10 gene knockout mice.

[0136] The above descriptions are merely embodiments of the present invention. Commonly known technical knowledge in the solutions is not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the filing date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can, under the guidance of this application, improve and implement this solution in combination with their own capabilities. Some typical well-known technologies should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. IL10 The application of gene editing in a pig model of inflammatory bowel disease is characterized by, The gene editing is selected from those capable of inactivating or reducing [the gene's] capacity. IL10 Gene expression is affected by gene knockout, gene knockdown, gene interference, or gene point mutation.

2. The application according to claim 1, characterized in that, The gene editing uses the CRISPR / Cas9 gene editing system, which specifically includes the following steps: (1) Extract pig genomic DNA, based on the pig's... IL10 Gene-designed specific amplification primers for amplification IL10 Genes, the ones mentioned IL10 The nucleotide sequence of the gene is as shown in SEQ ID NO:1 or has at least 90% identity with it; (2) According to the pig IL10 Gene-designed targeted pigs IL10 The sgRNA guide sequence of the gene, wherein the sgRNA guide sequence comprises any combination of two of the nucleotide sequences shown in SEQ ID NO.10-23; (3) The RNP complex formed by the sgRNA guide sequence and Cas9 protein described in step (2) was transfected into porcine fetal fibroblasts by electroporation. (4) Obtaining by flow cytometry sorting and gene identification. IL10 Gene-edited pig fetal fibroblasts.

3. The application according to claim 2, characterized in that, The specific amplification primers mentioned in step (1) are one of the primer pairs consisting of any two primers shown in the following sequence numbers: SEQ ID NO:2 and SEQ ID NO:3; SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7; SEQ ID NO:8 and SEQ ID NO:

9.

4. The application according to claim 3, characterized in that, The sgRNA guide sequence in step (2) is a combination of the nucleotide sequences shown in SEQ ID NO:13 and SEQ ID NO:

19.

5. The application according to claim 3, characterized in that, The gene editing also includes the following steps: (5) Preparation of reconstructed embryos: The embryos obtained in step (4) IL10 Gene-edited pig fetal fibroblasts were used as donor cells. Oocytes were collected and enucleated. Then, the donor cells and oocytes were recombined using somatic cell nuclear transfer technology to obtain reconstructed embryos. (6) Reconstructed embryo fusion activation: The reconstructed embryo is activated to obtain a developable embryo. IL10 Gene-edited pig embryos reconstructed; (7) Embryo transfer: The activated reconstructed embryo is transferred into the pig uterus for pregnancy, and the embryo is obtained. IL10 Gene-edited pigs.

6. The application according to claim 5, characterized in that, The obtained IL10 The method for identifying gene-edited pigs is as follows: genomic DNA is extracted from their ear margin tissue, and the specific amplification primers are used to amplify it. IL10 The target gene fragment is amplified by PCR and then identified.

7. The application according to claim 6, characterized in that, The PCR amplification obtained in the identification step IL10 If the gene electrophoresis band contains only one band corresponding to the size of the gene knockout band, the identification result is that the gene knockout homozygote is IL10. - / - If the electrophoresis strip shows two bands, one corresponding to the size of the gene knockout band and the other corresponding to the size of the wild-type band, then the identification result is a gene knockout heterozygote IL10. + / - .

8. An sgRNA guide sequence combination for use in any one of claims 1-7, characterized in that... The sgRNA guide sequence includes any combination of two of the nucleotide sequences shown in SEQ ID NO.10-23.

9. The sgRNA guide sequence combination according to claim 8, characterized in that, The sgRNA guide sequence is a combination of the nucleotide sequences shown in SEQ ID NO:13 and SEQ ID NO:

19.

10. A biomaterial, characterized in that... The biological material contains: 1) the sgRNA guide sequence combination of claim 8 or 9, and / or 2) one or more of a vector, expression cassette or gene-editing cell comprising the sgRNA guide sequence combination of claim 8 or 9.

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