Application of genes derived from rumen abdominal sac tissue of ruminants in promoting rumen epithelial stem cell differentiation

Abstract

This invention discloses the application of genes derived from the rumen abdominal sac tissue of ruminants in promoting the differentiation of rumen epithelial stem cells, belonging to the field of genetic engineering technology. This invention is the first to discover genes with unknown functions. ABCC1、NPC1L1 , MRPL24 or GPR15LG The proteins encoded by these substances promote the differentiation of rumen epithelial stem cells, and this can be achieved by regulating gene expression or utilizing their encoded proteins to regulate the proliferation and differentiation of rumen epithelial stem cells. The research results of this invention overcome the limitations of traditional breeding methods that rely on phenotypic selection, by screening… ABCC1、NPC1L1 , MRPL24 or GPR15LG This study provides a reference for individuals with high gene expression and for the targeted breeding of breeds with more efficient rumen function using biological methods, and provides a core target for the breeding of new ruminant breeds that are high-yielding, efficient, and stress-resistant.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of genes derived from the rumen abdominal sac tissue of ruminants in promoting the differentiation of rumen epithelial stem cells. Background Technology

[0002] The rumen is a unique and vital digestive and absorptive organ in ruminants, serving as the primary site for nutrient digestion and metabolism. Nutrients are absorbed, transported, and metabolized through the rumen epithelium to provide energy for the body. The differentiation of rumen epithelial stem cells plays a crucial role in maintaining animal health and improving feed efficiency, directly impacting growth performance, feed utilization, and overall health. Therefore, in-depth research into methods and related genes that promote rumen epithelial stem cell differentiation is of great significance for optimizing ruminant farming efficiency and ensuring animal health.

[0003] In recent years, with the rapid development of molecular biology techniques, research on rumen epithelial stem cells has made some progress. Advanced technologies such as single-cell sequencing have identified cell populations with stem cell characteristics within the rumen epithelium. Some studies have also discovered genes and signaling pathways related to rumen epithelial development. For example, the insulin-like growth factor 1 (IGF-1) signaling pathway plays a crucial role in the adaptation of the rumen epithelium to high-concentrate diets. However, the regulatory mechanisms of rumen epithelial stem cell differentiation are not yet fully understood, and many unknown genes and signaling pathways remain to be discovered. Discovering new genes that promote rumen epithelial stem cell differentiation and applying them to practical production holds promise for achieving precise regulation of rumen development and function, bringing new breakthroughs and development opportunities to the ruminant farming industry. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide the application of genes derived from the rumen abdominal sac tissue of ruminants in promoting the differentiation of rumen epithelial stem cells.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, this invention provides the application of a gene derived from the rumen abdominal sac tissue of a ruminant in the preparation of a product regulating the differentiation of rumen epithelial stem cells, wherein the gene derived from the rumen abdominal sac tissue of a ruminant is... ABCC1 NPC1L1 , MRPL24 or GPR15LG At least one of them.

[0007] The gene ABCC1, NPC1L1 , MRPL24 and GPR15LGThe gene accession numbers are XM_027961611, XM_004008262, XM_027976525 and XM_004021530, respectively.

[0008] The ABCC1 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0009] The NPC1L1 The nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0010] The MRPL24 The nucleotide sequence is shown in SEQ ID NO. 5, and the amino acid sequence is shown in SEQ ID NO. 6.

[0011] The GPR15LG The nucleotide sequence is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8.

[0012] Preferably, through overexpression ABCC1, NPC1L1 , MRPL24 or GPR15LG, To promote the differentiation of rumen epithelial stem cells; by inhibiting ABCC1, NPC1L1 , MRPL24 or GPR15LG Gene expression can delay the differentiation of rumen epithelial stem cells.

[0013] In a second aspect, this invention provides the application of a gene-encoded protein derived from the rumen abdominal sac tissue of ruminants in the preparation of a product regulating the differentiation of rumen epithelial stem cells, wherein the gene derived from the rumen abdominal sac tissue of ruminants is... ABCC1, NPC1L1 , MRPL24 or GPR15LG At least one of them.

[0014] Preferably, by enhancing ABCC1, NPC1L1 , MRPL24 or GPR15LG Activity of gene-encoded proteins ， To promote the differentiation of rumen epithelial stem cells; by inhibiting ABCC1, NPC1L1 , MRPL24 or GPR15LG The activity of gene-encoded proteins can delay the differentiation of rumen epithelial stem cells.

[0015] In a third aspect, the present invention provides a method for promoting the differentiation of rumen epithelial stem cells, the method comprising: causing rumen epithelial stem cells to... ABCC1, NPC1L1 , MRPL24 or GPR15LG At least one of the genes is overexpressed.

[0016] The promotion of rumen epithelial stem cell differentiation specifically manifests as: reducing the number of stem cells in rumen organoids and / or promoting the growth of rumen organoids.

[0017] The term "organoid" refers to a three-dimensional (3D) structural model derived from the organism's own tissues or stem cells and formed through in vitro 3D culture, exhibiting a high degree of similarity to the original tissues and organs in terms of tissue structure, cell type, and function. Currently, organoid models not only show significant application prospects in research such as human disease modeling, drug development, precision medicine, regenerative medicine, and biomaterials, but are also applied in research on nutritional regulation of ruminants in animal husbandry.

[0018] The term "rumen organoids" refers to three-dimensional (3D) rumen structural models derived from rumen tissue and formed through in vitro 3D culture, possessing the original tissues and organs. Studies have shown that rumen epithelial organoid models are helpful in studying the keratinization process of the rumen epithelium. When the organoids reach a size of 140 μm, they exhibit eosinophilic mature keratinocytes similar to the in vivo keratinocytes. Furthermore, the expression patterns of marker genes in organoids are similar to those in rumen papillae. Generally, the induction efficiency under different conditions is compared by the number of rumen epithelial organoids; a higher number of organoids indicates a higher success rate. The diameter reflects the degree of cell proliferation activity; a significantly larger diameter indicates good activity.

[0019] The term "organoid growth" refers to the increase in the volume, number of stem cells, or functional maturity of an already formed organoid, reflecting the ability of organoids to expand and mature.

[0020] The ABCC1, NPC1L1 , MRPL24 or GPR15LG The steps of gene overexpression are as follows:

[0021] (1) Insert the gene from the rumen sac tissue of ruminants into the V5 tag to obtain the V5 tag fusion gene, and clone the V5 tag fusion gene into the lentiviral transfer plasmid to obtain the recombinant lentiviral transfer plasmid.

[0022] (2) The recombinant lentivirus transfer plasmid obtained in step (1) was co-transfected with packaging plasmid and envelope plasmid into 293T cells, and the lentivirus supernatant was collected and concentrated.

[0023] (3) Infect rumen epithelial organoid single cells with the concentrated lentivirus supernatant obtained in step (2), screen for stably infected rumen epithelial organoid single cells, and achieve gene overexpression.

[0024] Preferably, in step (1), the lentivirus transfer plasmid is pLVX-EF1α; in step (2), the packaging plasmid is PSPAX2 and the envelope plasmid is pMD2G.

[0025] The beneficial effects of this invention are:

[0026] This invention is the first to discover genes with unknown functions. ABCC1, NPC1L1 , MRPL24 or GPR15LG The proteins encoded by these substances promote the differentiation of rumen epithelial stem cells, and this can be achieved by regulating gene expression or utilizing their encoded proteins to regulate the proliferation and differentiation of rumen epithelial stem cells. The research results of this invention overcome the limitations of traditional breeding methods that rely on phenotypic selection, by screening… ABCC1, NPC1L1 , MRPL24 or GPR15LG This study provides a reference for individuals with high gene expression and for the targeted breeding of breeds with more efficient rumen function using biological methods, and provides a core target for the breeding of new ruminant breeds that are high-yielding, efficient, and stress-resistant. Attached Figure Description

[0027] Figure 1 This represents the percentage of the three animal cell types in single-cell transcriptome analysis.

[0028] Figure 2 This represents the gene expression level of the target gene in the epithelial subtype of the single-cell transcriptome; whereby... Figure 2 a- Figure 2 The 'e' in the middle represents... GPR15LG, MRPL24, NRF1, NPC1L1 and ABCC1 Gene expression levels in epithelial subtypes.

[0029] Figure 3 This is a schematic diagram of a recombinant lentivirus transfer plasmid.

[0030] Figure 4 These are representative images of rumen epithelial organoids 10 days after infection with a GFP-expressing lentivirus; among them, Figure 4 a- Figure 4 In the image, c represents the bright-field image, the fluorescence image, and the image formed by combining the bright-field image and the fluorescence image, respectively.

[0031] Figure 5 The relative expression levels of six target genes in rumen organoids infected with lentiviruses.

[0032] Figure 6 Electron micrographs of positively transfected cells after 16 days of culture to form rumen epithelial organoids; Figure 6 a- Figure 6 The image shows the control group (EGFP). NRF1 , SLC22A14 , NPC1L1 , ABCC1 , GPR15LG and MRPL24 Electron micrographs of the gene were obtained because the experiment was conducted in multiple sessions, thus involving electron micrographs of three control groups (EGFP).

[0033] Figure 7 The statistical results of the number of rumen organoids formed from positively transfected cells after 16 days of culture; among them, Figure 7 a- Figure 7 c shows the control group (EGFP). NRF1 , SLC22A14 , NPC1L1 , ABCC1 , GPR15LG and MRPL24 The number of stem cells in rumen organoids formed by gene-transfected cells after 16 days of culture.

[0034] Figure 8 The statistical results of the diameter of rumen epithelial organoids formed from positively transfected cells after 16 days of culture; among them, Figure 8 a- Figure 8 c shows the control group (EGFP). NRF1 , SLC22A14 , NPC1L1 , ABCC1 , GPR15LG and MRPL24 Statistical results of rumen organoid diameter after 16 days of culture of gene-transfected cells forming rumen epithelial organoids.

[0035] Figure 9 The results show the expression levels of keratinization-related genes in rumen epithelial organoids formed from positively transfected cells after 16 days of culture; among them... Figure 9 a- Figure 9 c shows the control group (EGFP). NRF1 , SLC22A14 , NPC1L1 , ABCC1 , GPR15LG and MRPL24 Keratinization-related genes in rumen organoids cultured for 16 days after gene transfection into cells forming rumen epithelial organoids CNFN and TGM1 The amount of expression. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0037] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0038] Example 1: Screening of target genes

[0039] 1. Test Methods

[0040] Transcriptome sequencing and single-cell transcriptome sequencing were performed on rumen and abdominal sac tissues of ruminants, including roe deer, sika deer, and sheep. Single-cell transcriptome sequencing was used to segment and identify differentially expressed genes in each of the three animal groups.

[0041] (1) Transcriptome sequencing and analysis

[0042] RNA was extracted from rumen abdominal sac tissue for library construction and sequenced on the Illumina NovaSeq 6000 platform. Raw sequencing data were processed using Fastp v0.20.0, Scythe v0.991, and Sickle v1.33 software to remove adapter sequences and filter low-quality sequences. High-quality sequences preserved using HISAT2 v2.2.0 were aligned to a ruminant reference genome. Transcript abundance was calculated using StringTie v2.1.3b software and expressed as FPKM (number of transcript fragments per kilobase per million aligned sequences). Differentially expressed genes (DEGs) were identified using DESeq2 v1.42.1, with a selection criterion of |log2(foldchange)| ≥ 1 and p <0.05. Gene Ontology (GO) and KEGG pathway enrichment analyses of DEGs were performed using Metascape software.

[0043] (2) Single-cell transcriptome sequencing and analysis

[0044] The method for isolating cells from the rumen abdominal sac tissue was as follows: The rumen abdominal sac tissue was dissected, washed in cold HBSS buffer, and digested with 0.25% trypsin at 37°C for 25 minutes. The digested cells were filtered through a 40 μm filter, centrifuged at 1500 rpm (4°C), and resuspended in PBS. Cell viability and density were assessed using a Bio-Rad TC20 automated cell counter, adjusted to 1×10⁻⁶ cells / mL. 6Cells / mL. Single-cell RNA sequencing libraries were constructed using the Chromium Single Cell 3′ Kit v3.1 and sequenced on an Illumina NovaSeq 6000 platform. Raw data were unmixed, aligned, and quantified using Cell Ranger v7.1.0. Cross-species homologous genes were identified using DIAMOND v2.1.6.160. Cells expressing ≥200 genes and with mitochondrial gene expression <5% were retained. The datasets were merged, normalized (NormalizeData), and integrated (FindIntegrationAnchors and IntegrateData) using Seurat v3.1.4, and mitochondrial genes were removed. Principal component analysis (PCA) was performed using the Jackstraw permutation test to identify significant principal components. Cell clustering was performed using the FindClusters function (resolution=0.6) and visualized using UMAP. The criteria for identifying marker genes (FindAllMarkers) and differentially expressed genes (|log2(fold change)| ≥ 1 and...) were defined. p <0.05).

[0045] Cell clustering was initially performed using FindClusters in Seurat v3.1.4. Next, the FindAllMarkers function was used to identify differentially expressed genes (|log2(fold change)| ≥ 1) in single-cell transcriptomes. p <0.05).

[0046] 2. Test Results

[0047] Single-cell transcriptome analysis revealed that after cell grouping, epithelial cells were identified as basal cells, spinous cells, granular cells, and mitotic cells. Two subtypes were identified in basal cells: basal cell_PBP2+ and basal cell_TP63+. Two subtypes were identified in spinous cells: spinous cell_MET+ and spinous cell_SLC22A23+. Granular cells were identified as the granular cell_CLDN4+ subtype. Figure 1 ).

[0048] Single-cell transcriptome analysis revealed that in the roe deer's predominantly proliferation-related cell subtypes (mitotic cells and basal cells_TP63+), transcription factors... NRF1 ,Gene MRPL24 and GPR15LG High expression (|log2(fold change)| ≥ 1.5, p <0.05)( Figure 2Further transcriptomic analysis revealed differentially expressed genes ( ) in the rumen of roe deer. SLC22A14 , NPC1L1 , ABCC1 Enrichment of vitamin transport pathways (|log2(fold change)| ≥ 1, p <0.05 (Table 1)

[0049] Table 1. GO enrichment of differentially expressed genes in the transcriptomes of three animal species, focusing on genes involved in vitamin transport.

[0050]

[0051] The results of the above screening for genes that may be related to rumen cell differentiation are as follows: NRF1 (XM_060414825) SLC22A14 (XM_027958019) NPC1L1 (XM_004008262) ABCC1 (XM_027961611) GPR15LG (XM_004021530) and MRPL24 (XM_027976525).

[0052] Example 2: Application of overexpression of target gene in rumen epithelial stem cell differentiation.

[0053] 1. Test Methods

[0054] (1) Culture of rumen epithelial organoids

[0055] Rumen tissue was obtained from 7-day-old sheep, and the muscle layer was manually dissected. The epithelial tissue was digested with trypsin at 37°C for 30 minutes. The digested cell suspension was filtered through a 70 μm cell sieve, and the isolated single cells were centrifuged at 1000 rpm for 3 minutes to remove trypsin residue. The cells were resuspended in organoid culture medium at a density of 50–100 cells / μL, mixed with an equal volume of extracellular matrix (Vazyme, GL101), and seeded at 50 μL per well. After curing at 37°C for 30 minutes, organoid culture medium was added, and the medium was changed every 48 hours until the end of the experiment, and cultured for 12 days.

[0056] (2) Plasmid construction and lentivirus packaging

[0057] Obtained from the NCBI database NRF1 , SLC22A14 , NPC1L1 , ABCC1 , GPR15LG and MRPL24 The open reading frame (ORF) sequence of a gene. NRF1 , SLC22A14 , NPC1L1 , ABCC1 , GPR15LG and MRPL24 V5 tags that maintain the reading frame are inserted before the stop codon of the gene, a process synthesized by GenCEFE®.

[0058] The V5 tag has been integrated NRF1 , SLC22A14 , NPC1L1 , ABCC1 , GPR15LG and MRPL24 The gene was subcloned into a plasmid vector (pLVX-EF1α), resulting in six recombinant lentiviral transfer plasmids. Figure 3 This is a schematic diagram of a recombinant lentiviral transfer plasmid. The EGFP part in the diagram can be replaced with the ORF sequence of different genes.

[0059] Six types of lentiviral packaging particles were obtained by co-transfecting 293T cells with recombinant lentiviral transfer plasmids, packaging plasmids, PSPAX2, and pMD2G plasmids. Viral supernatants were collected at 48 and 72 hours after transfection with the six types of lentiviral packaging particles and concentrated using lentiviral precipitation buffer (Servicebio, # G1801) according to the manufacturer's instructions.

[0060] (3) Lentiviral infection

[0061] Lentiviral transduction was performed on organoids cultured for 72 hours. First, organoids were dissociated into single cells using TrypLE™ (Gibco, #12604013). Cells were infected for 6 hours at an MOI of 10. After infection, residual viral particles were removed by centrifugation, and the cells were then re-encapsulated in extracellular matrix and cultured for 48 hours. Selection was then performed by adding 1 μg / mL puromycin until all uninfected control organoids were completely eliminated.

[0062] (4) Determination of organoid size and number

[0063] An image of one region within the field of view of each extracellular matrix-embedded organoid was randomly selected for imaging. The images of the four organoids were then analyzed using ImageJ software, and the size and number of organoids were quantified using the "Analyze > Tools > ROI Manager" function.

[0064] (5) RNA extraction and real-time quantitative PCR

[0065] Matrigel cells were dissolved in ice-cold PBS solution, cultured, and collected. Cells were then recovered by low-speed centrifugation. Total RNA was extracted using a microRNA extraction kit (Magen, #R4012). Reverse transcription was performed using HiScript II Q RT SuperMix (Vazyme, #R223), and real-time quantitative PCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme, #Q111).

[0066] (6) Data Analysis

[0067] The significance of the above analysis p A value <0.05 was considered statistically significant. For validation of gene overexpression in rumen epithelial organoids, the statistical analysis employed an unpaired two-tailed Student's t-test.

[0068] 2. Test Results

[0069] (1) Representative images of rumen epithelial organoids 10 days after infection with GFP-expressing lentivirus were shown as follows: Figure 4 As shown, Figure 4 The image includes three sets of images: Bright: This shows a bright-field image of the organoid, revealing its morphology and structure. EGFP (Enhanced Green Fluorescent Protein): This image, captured using a fluorescence microscope, shows GFP expression; the green fluorescence represents cells in the organoid that successfully express GFP, and the distribution of these cells is shown. Merge: This image combines the bright-field and fluorescence images to help better identify the structure of GFP-expressing cells and organoids; the presented images demonstrate that the organoid successfully expresses the gene derived from the rumen sac tissue of ruminants.

[0070] (2) Real-time quantitative PCR analysis to verify the target gene in rumen organoids infected with lentivirus. NRF1 , SLC22A14 , NPC1L1 , ABCC1 , GPR15LG and MRPL24 The relative expression levels of the six genes indicate that real-time quantitative PCR analysis verified the successful overexpression of the target genes in lentivirally infected rumen organoids. Figure 5 ).

[0071] (3) Electron micrographs of positively transfected cells after 16 days of culture to form rumen epithelial organoids showed that: overexpression NPC1L1 , ABCC1 , GPR15LG and MRPL24 Four genes resulted in hyperkeratotic organoids. Figure 6 ).

[0072] (4) The statistical results of the number of rumen organoids formed by positively transfected cells after 16 days of culture showed that: overexpression NPC1L1 , ABCC1 , GPR15LG and MRPL24 The number of organoids in the overexpressing group was significantly lower than that in the control group (EGFP), while the number of organoids in the overexpressing group was significantly lower. NRF1 and SLC22A14 The number of organoids decreased compared to the control group, but the difference was not statistically significant. Figure 7 The rumen epithelium is a typical stratified squamous epithelium, similar to organoids of the esophagus and vagina. Initially, basal cells with stem cell characteristics proliferate to form stem cell clusters. Because the matrix, representing the cell matrix, encloses these clusters, the newly proliferating stem cells grow inward and lose their stem cell nature, gradually differentiating into different cells representing the four layers of the rumen epithelium. Therefore, a decrease in the number of organoids suggests overexpression. NPC1L1 , ABCC1 , GPR15LG and MRPL24 It weakened the stemness of rumen epithelial stem cells.

[0073] (5) The statistical results of the diameter of rumen organoids formed by positively transfected cells after 16 days of culture showed that: overexpression NPC1L1 , ABCC1 , GPR15LG and MRPL24 The diameter of rumen organoids was significantly increased compared to the control group (EGFP), while overexpression... NRF1 and SLC22A14 The diameter of the organoids did not change significantly compared to the control group (EGFP). Figure 8 The increase in organoid diameter indicates... NPC1L1 , ABCC1 , GPR15LG and MRPL24 Overexpression of the gene promoted the growth of rumen organoids.

[0074] (6) CNFN and TGM1 The gene is a keratinization-related gene. Keratinization is a marker of rumen epithelial cell maturation and final developmental fate, reflecting the differentiation level of rumen epithelial stem cells. The expression levels of the keratinization-related gene in rumen organoids formed from positively transfected cells after 16 days of culture showed overexpression. NPC1L1 , ABCC1 , GPR15LG and MRPL24 The expression levels of keratinization-related genes in rumen organoids were significantly increased, indicating that... NPC1L1 , ABCC1 , GPR15LG and MRPL24Overexpression of this substance promotes the differentiation of rumen epithelial stem cells. Figure 9 ).

[0075] In summary, ABCC1 , NPC1L1 , MRPL24 and GPR15LG Overexpression of the gene reduced the number of stem cells in rumen organoids, promoted the growth of rumen organoids, and promoted the differentiation of rumen epithelial stem cells.

[0076] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted.

[0077] Equivalent substitutions, improvements, etc., should all be included within the scope of protection of this application.

Claims

1. Use of a gene derived from the rumen omental tissue of a ruminant for the preparation of a product for regulating the differentiation of rumen epithelial stem cells, characterized in that, The gene derived from the rumen omental tissue of a ruminant is at least one of ABCC1, NPC1L1 , MRPL24 or GPR15LG .

2. Use according to claim 1, characterized in that, The ABCC1, NPC1L1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. MRPL24 The nucleotide sequence of the gene is shown in SEQ ID NO.

3. GPR15LG The nucleotide sequence of the gene is shown in SEQ ID NO.

5. ABCC1, NPC1L1 The nucleotide sequence of the gene is shown in SEQ ID NO.

7.

3. Use according to claim 1, characterized in that, promote rumen epithelial stem cell differentiation by overexpressing MRPL24 , GPR15LG or ABCC1, NPC1L1 .

4. Use of a protein encoded by a gene derived from the tissue of the rumen omental bursa of a ruminant for the preparation of a product for regulating the differentiation of rumen epithelial stem cells, characterized in that, The gene derived from the rumen omental tissue of a ruminant is at least one of MRPL24 , GPR15LG or ABCC1, NPC1L1 .

5. Use according to claim 4, characterized in that, By enhancing MRPL24 , GPR15LG or ABCC1, NPC1L1 Activity of gene-encoded proteins ， To promote the differentiation of rumen epithelial stem cells.

6. A method of promoting differentiation of rumen epithelial stem cells, characterized by, The method is to overexpress at least one gene in the rumen epithelial stem cell in MRPL24 , GPR15LG or The promoting rumen epithelial stem cell differentiation specifically manifests as: reducing the number of stem cells in the rumen organoid and / or promoting the growth of the rumen organoid. .

7. The method of claim 6, wherein, ABCC1, NPC1L1 8. The method of claim 6, wherein, make MRPL24 , GPR15LG or (1) inserting a gene derived from rumen abdominal sac tissue of a ruminant into a V5 tag to obtain a V5 tag fusion gene, and cloning the V5 tag fusion gene into a lentivirus transfer plasmid to obtain a recombinant lentivirus transfer plasmid; The steps of gene overexpression are as follows: (2) co-transfecting the recombinant lentivirus transfer plasmid obtained in step (1) with a packaging plasmid and an envelope plasmid into 293T cells, collecting lentivirus supernatant, and concentrating the lentivirus supernatant; (3) infecting rumen epithelial organoid single cells with the concentrated lentivirus supernatant obtained in step (2), screening for stably infected rumen epithelial organoid single cells, and realizing gene overexpression. In step (1), the lentivirus transfer plasmid is pLVX-EF1a; in step (2), the packaging plasmid is PSPAX2, and the envelope plasmid is pMD2G.

9. The method of claim 8, wherein, ​

Images