Sgrna molecules targeting igfbp3 gene for knockdown or knockout and application thereof in increasing body weight of deer

Abstract

The application discloses an sgRNA molecule for knocking down or knocking out an IGFBP3 gene and application thereof in increasing deer antler weight, and relates to the technical field of deer antler regeneration. Knocking out the IGFBP3 gene in the caprine horn shank periosteum cells can promote the proliferation and differentiation of deer antler stem cells, inhibit cell apoptosis, and reduce the cell apoptosis rate. Promoting the proliferation of the deer antler stem cells is helpful to increasing the deer antler weight. The verification of the main function of the IGFBP3 gene provides more accurate and effective data for molecular selection and breeding of high-yield caprine. Therefore, the inhibitor of the IGFBP3 gene and / or the inhibitor of IGFBP3 protein are expected to increase the deer antler weight, and have a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of deer antler regeneration technology, and more specifically, to sgRNA molecules that target and knock down or eliminate the IGFBP3 gene and their application in increasing deer antler weight. Background Technology

[0002] Sika deer antlers are non-ossified, hairy young antlers derived from male sika deer (Cervus nippon Temminck). As a precious traditional animal medicine, they have been widely used in China for over 2000 years. Antlers are the only mammalian organ that can rapidly regenerate annually without becoming cancerous, making them an ideal model for regeneration research. In China, the number of farmed sika deer used for antler production is increasing year by year. Therefore, increasing antler production has become a key objective for sika deer farming.

[0003] The growth of deer antlers is mainly influenced by factors such as genetics, nutritional conditions, and hormone levels. Insulin-like growth factor-1 (IGF-1) is a peptide growth factor that promotes the proliferation and differentiation of antler chondrocytes. Insulin-like growth factor binding protein 3 (IGFBP3) is one of the six members of the IGFBP family and is the most important carrier protein of IGF-1. It regulates cellular function through both IGF-1-dependent mechanisms (involving IGF-1 signaling) and independent mechanisms (not involving IGF-1 signaling).

[0004] Currently, the genes associated with the weight of sika deer antlers are still unclear.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide the application of inhibitors of the IGFBP3 gene and / or IGFBP3 protein in increasing the weight of deer antlers and methods for increasing the weight of deer antlers, thereby providing favorable technical support for increasing the yield of deer antlers.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides an sgRNA molecule that targets and knocks down or eliminates the IGFBP3 gene, wherein the target sequence of the IGFBP3 gene targeted by the sgRNA is shown in at least one of SEQ ID NO: 1-8.

[0009] In a preferred embodiment of the present invention, the nucleotide sequence of the sgRNA molecule is shown in at least one of SEQ ID NO: 9-12.

[0010] Secondly, the present invention provides a nucleic acid molecule that encodes the aforementioned sgRNA molecule that targets and knocks down or eliminates the IGFBP3 gene.

[0011] Thirdly, the present invention provides the application of sgRNA molecules or the above-mentioned nucleic acid molecules that target and knock down or eliminate the IGFBP3 gene in knocking down or eliminating the IGFBP3 gene in cells.

[0012] Fourthly, the present invention provides a method for knocking down or knocking out the IGFBP3 gene in cells, comprising: knocking down or knocking out the IGFBP3 gene using the above-mentioned sgRNA molecule that targets and knocks down or knocks out the IGFBP3 gene.

[0013] In a preferred embodiment of the present invention, when knocking down or knocking out the IGFBP3 gene, the gene editing tool used is selected from any one of CRISPR / Cas9, CRISPR / Cas13, CRISPR / Cas12, and CRISPR / Cas14.

[0014] In a preferred embodiment of the present invention, the method for knocking down or eliminating the IGFBP3 gene includes: infecting monoclonal deer antler stem cells with an sgRNA-Cas protein recombinant vector to achieve the knockout of the IGFBP3 gene.

[0015] Fifthly, the present invention provides the application of an inhibitor of the IGFBP3 gene and / or IGFBP3 protein in increasing the weight of deer antlers.

[0016] Sixthly, the present invention also provides the application of an inhibitor of the IGFBP3 gene and / or an inhibitor of the IGFBP3 protein in the proliferation of deer antler stem cells.

[0017] In a seventh aspect, the present invention also provides a method for increasing the weight of deer antlers, comprising: causing the IGFBP3 gene in deer to be either not expressed or suppressed.

[0018] The present invention has the following beneficial effects:

[0019] This invention uses genome-wide association analysis (GWAS) to annotate the candidate gene IGFBP3 associated with sika deer antler weight. Experiments show that the IGFBP3 gene plays a crucial regulatory role in sika deer antler stalk periosteum cells (PPCs). Knocking out the IGFBP3 gene in sika deer antler stalk periosteum cells using CRISPR-Cas9 technology promotes the proliferation and differentiation of antler stem cells, inhibits apoptosis, and reduces the apoptosis rate. Promoting antler stem cell proliferation helps increase antler weight. Validation of the main function of the IGFBP3 gene provides more precise and effective data for the molecular breeding of high-yielding sika deer. Therefore, inhibitors of the IGFBP3 gene and / or IGFBP3 protein hold promise for increasing antler weight, which will improve the accuracy and efficiency of high-yielding sika deer breed selection. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The results of genome-wide association analysis for the antler weight trait are shown. A: Manhattan plot of the significance of SNPs associated with antler weight trait, with the dashed line representing the significance threshold; B: Quantile-quantile plot;

[0022] Figure 2 The images show the enzyme digestion electrophoresis diagram (A) and sequencing alignment results (B) of the recombinant plasmid after construction.

[0023] Figure 3 For IGFBP3 - / - Image showing the results of gene knockout effect identification in cell lines (A: IGFBP3) - / - A: Cell sequencing identification results; B: Western blotting detection of IGFBP3 gene knockout effect; C: IGFBP3 - / - Cellular IGFBP3 protein expression. "*" indicates WT vs. IGFBP3. - / - , P<0.05, "#": Vector VS IGFBP3 - / - (P<0.05);

[0024] Figure 4 For IGFBP3 - / - Experimental results on the effect of PI staining on PPC cell proliferation (A to D: PI staining detection of IGFBP3) - / -Effects on PPC cell cycle; D: CCK8 assay for IGFBP-3 - / - Effects on PPC cell proliferation;

[0025] Figure 5 For IGFBP3 - / - Figure 1 shows the experimental results regarding the effect of Annexin V-PI double staining on PPC cell apoptosis. A to D: Detection of IGFBP3 by Annexin V-PI double staining. - / - Effects on PPC cell apoptosis; E: IGFBP3 - / - Statistics on the total apoptosis rate of PPC cells; "*" indicates WTVS IGFBP3. - / - , P<0.05, "#": Vector VS IGFBP3 - / - (P<0.05). Detailed Implementation

[0026] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0027] Antler weight is the most important heritable trait in molecular breeding of sika deer. Therefore, clarifying the functions of key candidate genes regulating antler weight is beneficial to improving the accuracy and efficiency of breeding high-yielding sika deer breeds. In particular, by knocking out the IGFBP3 gene in the periosteum cells of sika deer antler stalks, the proliferation inhibition and apoptosis promotion effects of IGFBP3 on antler stem cells were discovered. The determination of the main function of the IGFBP3 gene provides more precise and effective data for the molecular breeding of high-yielding sika deer. Inhibitors of the IGFBP3 gene are expected to increase antler weight, which will help improve the accuracy and efficiency of breeding high-yielding sika deer breeds.

[0028] In a first aspect, the present invention provides an sgRNA molecule that targets and knocks down or eliminates the IGFBP3 gene, wherein the target sequence (or target sequence) of the IGFBP3 gene targeted by the sgRNA is shown in at least one of SEQ ID NO: 1-8.

[0029] Table 1. Target Sequences

[0030]

[0031] In a preferred embodiment of the present invention, the crRNA nucleotide sequence of the sgRNA molecule is shown in at least one of SEQ ID NO: 9-12:

[0032] SEQ ID NO: 9:

[0033] CGGCUCGUGCCGCAGGCGG. Target sequence 1.

[0034] SEQ ID NO: 10: ACGGCUCGCACGCACCACC. Target sequence 2.

[0035] SEQ ID NO: 11: CGCUCGGUGUAGACGCCGCA. Target sequence 3.

[0036] SEQ ID NO: 12: AUGGCUGUGGUCUUCUUCCG. Target sequence 4.

[0037] In a preferred embodiment of the present invention, the sgRNA molecule further includes a scaffold sequence operatively linked to the crRNA sequence, the specific sequence of which is as follows:

[0038] 5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCU AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3', SEQ ID NO: 13.

[0039] Secondly, the present invention provides a nucleic acid molecule that encodes the aforementioned sgRNA molecule that targets and knocks down or eliminates the IGFBP3 gene.

[0040] Thirdly, this invention provides the application of sgRNA molecules that target and knock down or eliminate the IGFBP3 gene in knocking down or eliminating the IGFBP3 gene in cells.

[0041] Fourthly, the present invention provides a method for knocking down or knocking out the IGFBP3 gene in cells, comprising: knocking down or knocking out the IGFBP3 gene using the above-mentioned sgRNA molecule that targets and knocks down or knocks out the IGFBP3 gene.

[0042] In a preferred embodiment of the present invention, when knocking down or knocking out the IGFBP3 gene, the gene editing tool used is selected from any one of CRISPR / Cas9, CRISPR / Cas13, CRISPR / Cas12, and CRISPR / Cas14.

[0043] In a preferred embodiment of the present invention, the method for knocking down or eliminating the IGFBP3 gene includes: infecting monoclonal deer antler stem cells with an sgRNA-Cas protein recombinant vector to achieve the knockout of the IGFBP3 gene.

[0044] Fifthly, the present invention provides the application of an inhibitor of the IGFBP3 gene and / or IGFBP3 protein in increasing the weight of deer antlers.

[0045] The aforementioned measures to increase the weight of deer antlers include, but are not limited to: accelerating the growth and development of deer antler tissue, increasing the weight of deer antlers, and shortening the deer antler production cycle.

[0046] In a preferred embodiment of the present invention, the inhibitor of the IGFBP3 gene is a substance that can inhibit the level of the IGFBP3 gene in cells and / or tissues, and the inhibitor of the IGFBP3 protein is a substance that can inhibit the level or activity of the IGFBP3 protein in cells and / or tissues.

[0047] In a preferred embodiment of the present invention, the inhibitor of the IGFBP3 gene is a substance that can inhibit the level of the IGFBP3 gene in deer antler stem cells, and the inhibitor of the IGFBP3 protein is a substance that can inhibit the level or activity of the IGFBP3 protein in cells and / or tissues.

[0048] In a preferred embodiment of the present invention, the deer antler stem cells are deer antler regeneration stem cells;

[0049] In a preferred embodiment of this invention, the antler regeneration stem cells are selected from antler stalk periosteum cells. Antler stalk periosteum cells belong to mesenchymal stem cells. Cartilage formation is driven by the proliferation and differentiation of mesenchymal stem cells into chondrocytes. Experimental results of this invention show that the IGFBP3 gene is an inhibitory factor for chondrocyte differentiation, and IGFBP3-induced apoptosis of antler stem cells may ultimately inhibit antler cartilage formation, thereby increasing antler weight.

[0050] In a preferred embodiment of the present invention, a substance that inhibits the level of IGFBP3 gene and / or the level or activity of IGFBP3 protein in cells of the sensitized area of ​​the horn stalk periosteum.

[0051] In a preferred embodiment of the present invention, the inhibitor of the IGFBP3 gene is selected from at least one of the following molecules, whether modified or unmodified:

[0052] siRNA, sgRNA and Cas proteins, snoRNA, shRNA and microRNA.

[0053] In a preferred embodiment of the present invention, the modification is a chemical modification, selected from one or more of methoxy modification, fluorination modification, thiophosphate modification, LNA modification, and methoxyethyl modification. Modification helps to improve the stability of nucleic acid molecules.

[0054] In a preferred embodiment of the present invention, the inhibitor of the IGFBP3 gene includes an sgRNA expression vector and a Cas protein expression vector, wherein the Cas protein is selected from any one of the following proteins: Cas9, Cas13a, Cas12 and Cas14.

[0055] In a preferred embodiment of the present invention, the sgRNA is an sgRNA that targets the first and second exons of the IGFBP3 gene.

[0056] In a preferred embodiment of the present invention, the sgRNA targets at least one of the sequences shown in SEQ ID NO: 1-8.

[0057]

[0058] Sixthly, this invention also provides the application of an inhibitor of the IGFBP3 gene in the proliferation of deer antler stem cells. The inventors have discovered that inhibiting the expression of the IGFBP3 gene helps reduce the number of cells in the G0 / G1 and G2 / M phases, promotes the proliferation of deer antler stem cells, and thus increases the weight of deer antlers.

[0059] In a preferred embodiment of the present invention, the deer antler stem cells are deer antler regeneration stem cells;

[0060] In a preferred embodiment of the present invention, the deer antler regeneration stem cells are selected from horn stalk periosteum cells;

[0061] In a preferred embodiment of the present invention, the inhibitor of the IGFBP3 gene is selected from at least one of the following molecules, whether modified or unmodified:

[0062] siRNA, sgRNA and Cas proteins, snoRNA, shRNA and microRNA.

[0063] In a seventh aspect, the present invention also provides a method for increasing the weight of deer antlers, comprising: causing the IGFBP3 gene in deer to be either not expressed or suppressed.

[0064] In a preferred embodiment of the present invention, gene knockout is used to suppress or eliminate the expression of the IGFBP3 gene in deer.

[0065] In a preferred embodiment of the present invention, gene knockout is either complete gene knockout or conditional gene knockout.

[0066] In a preferred embodiment of the present invention, the exon sequence of the IGFBP3 gene in deer is knocked out by gene knockout.

[0067] In a preferred embodiment of the present invention, at least one exon sequence of the first and second exons of the IGFBP3 gene is knocked out.

[0068] In a preferred embodiment of the present invention, the gene editing technology used for gene knockout is selected from at least one of the following: LEAPER technology, CRISPR / Cas gene editing technology, ZFN technology, TALEN technology, and Cre-loxp gene knockout technology.

[0069] LEAPER technology is a technique that introduces an RNA (arRNA) sequence complementary to the target site into the cell, recruiting ADAR proteins to edit the RNA at the target site. The RNA used in this technique, complementary to the target site sequence, can stimulate highly efficient RNA editing at the target site without any modification.

[0070] In a preferred embodiment of the present invention, the editing tool used in the CRISPR / Cas gene editing technology is selected from any one of CRISPR / Cas9, CRISPR / Cas13, CRISPR / Cas12, and CRISPR / Cas14.

[0071] In a preferred embodiment of the present invention, the gene knockout method includes: infecting monoclonal deer antler stem cells with an sgRNA-Cas protein recombinant vector to achieve the knockout of the IGFBP3 gene.

[0072] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0073] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0074] Material:

[0075] Pedicle periosteal cells (PPCs) and 293T cells were provided by the Pedicle Stem Cell Team of the Institute of Special Agricultural Products, Chinese Academy of Agricultural Sciences. Lenti-Guide-Puro plasmid was purchased from Changsha Fenghui Biotechnology Co., Ltd., and Stbl3 competent cells and Trans1-T1 competent cells were purchased from Beijing TransGen Biotech Co., Ltd. Cell culture was prepared using DMEM (Gibco, C11995500BT), FBS (Sigma-Aldrich, F8318), trypsin (Gibco, 25200056), antibiotics (Gibco, 15140-122), cell cryopreservation solution (Beyotime, C0210), and PBS (Beyotime, C0221A). Plasmids were prepared using the Beijing Tiangen Technology Plasmid Mini-Prep Kit (DP103), the Endotoxin-Free Plasmid Large-Scale Prep Kit (DP117), and a standard agarose gel DNA recovery kit.

[0076] Example 1

[0077] This embodiment provides an experimental method for GWAS annotation to correlate IGFBP3 with sika deer antler weight.

[0078] Genome-wide association study methods:

[0079] In GWAS analysis, individual kinship and population stratification are the main factors causing false positive associations. Therefore, a mixed linear model was used for trait association analysis, with population genetic structure as a fixed effect (the first three columns of PCA as population structure fixed effects) and individual kinship as a random effect to correct for the influence of population structure and individual kinship. GEMMA software was used for analysis, and the specific model is as follows:

[0080] y = Xα + Zβ + Wμ + e

[0081] y represents the phenotypic trait, X represents the indicator matrix of the fixed effects, α represents the estimated parameters of the fixed effects, Z represents the indicator matrix of the SNP, β represents the effect of the SNP, W represents the indicator matrix of the random effects, μ represents the predicted random individuals, and e represents the random residuals, which follow the pattern e ~ (0, δe²).

[0082] Using existing genome annotation results, gene function annotation was performed on significant SNP sites based on LD results, and enrichment analysis was conducted on the GO and KEGG databases.

[0083] result:

[0084] Genome-wide association analysis of the antler weight trait was performed using GEMMA software, resulting in a Manhattan diagram. Figure 1 (Image A in the text) and QQ image ( Figure 1(See Figure B in the diagram). The actual p-values ​​in the QQ plot deviate only slightly from the null hypothesis p-value in the tail region, indicating that trait differences are not caused by population stratification. GWAS results showed that 9 SNPs reached a significant level (p < 1e-6), see [see figure B in the diagram]. Figure 1 The IGFBP3 gene is located 22.8 kb away from the g.27759264A>G site (see Table 2).

[0085] Table 2. Annotation results of significant SNPs for antler weight traits.

[0086]

[0087] Example 2

[0088] This embodiment describes the construction and identification of recombinant plasmids.

[0089] 1. gRNA design, synthesis, and construction of recombinant plasmids

[0090] Based on the first and second exon sequences of the IGFBP3 sequence in the existing sika deer genome, four target sequences (target sequences 1 / 2 / 3 / 4) were designed using the CRISPR design website (hppt: / / zlab.bio / guide-design-resources) (Table 1). The DNA coding sequences of the designed sgRNAs (Table 2) were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0091] Table 1. Target Sequences

[0092]

[0093] Table 2. sgRNA sequences

[0094]

[0095] The Lenti-Guide-Puro plasmid was digested with BsmBI restriction enzyme at 4℃, and the digested plasmid was recovered by electrophoresis and gel extraction. Figure 2 (A) The annealed sgRNA DNA coding sequences shown in Table 2 were diluted 200-fold on an ice box to obtain the insert fragment solution. The reaction system was prepared on an ice box according to Table 2, mixed by pipetting, centrifuged, and then incubated at room temperature for 15 min. Subsequently, the mixture was transformed into Stbl3 competent cells, plated onto LB solid culture medium, and incubated overnight at 37°C. Single colonies were picked and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing verification. The successfully sequenced recombinant vector was subjected to plasmid extraction and stored at -80°C for later use. Sequencing results showed that the DNA coding fragments of all four sgRNAs were inserted into the corresponding positions of the vector, and the recombinant plasmid was successfully constructed. Figure 2 (B)

[0096] Table 2. DNA coding sequence of sgRNA and ligation system with linear vector

[0097]

[0098] Example 3

[0099] Identification of gene knockout effect in IGFBP3 cell line.

[0100] The experimental method is as follows:

[0101] 1. Cell infection

[0102] Lentiviral cells were thawed at 4°C and prepared for use. Transfection complexes were prepared according to the following groups: g1 / 2 / 3 / 4 recombinant vector group (5 μg / mL polybrene medium + recombinant vector virus), g0 empty vector group (5 μg / mL polybrene medium + empty vector), and blank group (5 μg / mL polybrene medium). After 12 hours of incubation, the medium was replaced with completely fresh medium, and culturing continued. 72 hours after infection, the original medium was discarded, and replaced with a mixed complete medium containing 5 μg / mL cymoxanil and 4 μg / mL puromycin. Double-drug screening was performed on infected cells. The blank group served as a control. After complete cell death in the blank group, the recombinant vector group and the empty vector group were cultured for another 96 hours. Cells were passaged when confluence reached over 85%.

[0103] 2. Selection and mutation detection of PPC monoclonal cell lines

[0104] Cells selected using dual-drug therapy were digested and counted with trypsin, then seeded at a density of 1000 cells / well in six-well plates to maintain cell suspension. Under a microscope, single suspended cells were aspirated using a micropipette and seeded into 96-well plates. 100 μL of complete culture medium containing 20% ​​FBS was added, and the plates were returned to a 37°C, 5% CO2 incubator for further culture to obtain monoclonal cell lines. Once cells had grown into colonies, they were digested with trypsin and transferred to 24-well plates for further culture. DNA was extracted from wild-type PPCs and lentivirally infected PPCs according to the DNA extraction kit instructions and stored at -20°C. PCR amplification of DNA fragments: Using wild-type and monoclonal cell line DNA as templates, high-fidelity DNA polymerase was used with primers to amplify DNA fragments containing Cas9 protein cleavage targets. The target DNA fragment was recovered, and its concentration was measured using a UV spectrophotometer. Following the kit instructions, the DNA fragment was gently mixed with the pEASY cloning vector in the correct ratio. After ligation, the product was transformed into Trans-T1 competent cells according to the transformation procedure. IPTG and X-gal were evenly spread on LB solid medium (containing AMP) and incubated at 37°C for 30 min to promote IPTG and X-gal uptake. Subsequently, 50 μL of bacterial culture was evenly spread on LB solid medium and incubated at 37°C for 12 h. White single colonies were picked and inoculated into 5 ml of LB liquid medium (containing AMP), incubated at 180 rpm at 37°C for 12 h, and then sent to Shanghai Sangon Biotech for sequencing.

[0105] 3. Western blotting detection of IGFBP3 protein expression

[0106] When the density of IGFBP3 KO, wild-type (WT) PPC, and PPC infected with empty vector lentivirus (Vector) reached 85% or higher, total protein was extracted, transferred to a PVDF membrane after polyacrylamide gel electrophoresis, blocked with antigen for 1 h, incubated with primary antibody overnight at 4°C, incubated with secondary antibody for 1 h, and then imaged using a protein imaging system (Tanon 5800).

[0107] The recombinant plasmid was co-transfected into PPCs using the method described above, and single-clone cell culture was performed. DNA was extracted from the single-clone cells, and the target fragment was amplified by PCR. After ligation with the pEASY vector, sequencing revealed that the single-cell survival rate was highest with G3 lentivirus infection, and three mutation types were observed. Western blot analysis of IGFBP3 protein expression in the three mutant cell types showed that mutant IGFBP3 protein was completely knocked out, confirming the successful construction of the IGFBP3 gene knockout single-clone cell line. Figure 3(A and B). After comparing the sequencing results of mutant 2 with the complete IGFBP3 genome sequence, it was found that the monoclonal cell line lacked 83 bases in the IGFBP3 allele. Figure 3 (C)

[0108] Example 4

[0109] Effects of IGFBP3 gene knockout on cell proliferation and apoptosis.

[0110] The experimental method is as follows:

[0111] 1. Propidium iodide (PI) DNA staining method for cell cycle detection

[0112] Wild-type cells, empty vector transfected cells, IGFBP3 - / - Cells were collected when the cell confluence reached 85%, centrifuged at 1000 rpm for 5 min to obtain a cell pellet, and fixed with 70% ethanol at 4°C for 24 h. After fixation, 500 μL of propidium iodide staining solution was added to each cell sample tube, and the cell pellet was slowly and thoroughly resuspended. The cells were then incubated at 37°C in the dark for 30 min. Flow cytometry was used to detect red fluorescence at an excitation wavelength of 488 nm, and light scattering was also measured.

[0113] 2. CCK8 assay for cell proliferation rate

[0114] Wild-type cells, empty vector transfected cells, IGFBP3 - / - Cells were cultured to the logarithmic growth phase and passaged into 96-well plates. Cells were counted using a cell counter, and 2000 cells were seeded per well. Cells were incubated at 37°C and 5% CO2 for 24 hours. The medium was then replaced with complete culture medium (10% FBS) containing 10% CCK8 reagent, and the cells were cultured for another 3 hours. Absorbance was measured at 450 nm using a microplate reader. This procedure was repeated every 24 hours, obtaining absorbance data at 24h, 48h, 72h, and 96h. Statistical analysis was performed on the data.

[0115] 3. Annexin V-PI double staining method for detecting cell apoptosis

[0116] Wild-type cells, empty vector transfected cells, IGFBP3 - / - Cells were harvested when cultured to 85% confluence, centrifuged at 1000 rpm for 5 min, and the cell pellet was resuspended in 500 μL of 1x Binding Buffer. 5 μL of Annexun V-FITC and 5 μL of Propidium lodide were added, gently mixed, and incubated at room temperature in the dark for 20 min. A negative control group (i.e., normal cells cultured without Annexun V-FITC and Propidium lodide) was also established. Cells were then analyzed by flow cytometry.

[0117] 4. Statistical Analysis

[0118] Data was entered using Excel and analyzed using SPSS 22.0 statistical software. Data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant. GraphPad Prism 8.0 was used to create bar charts.

[0119] Cell cycle analysis showed that, compared with empty vector cells, IGFBP3 gene knockout reduced G0 / G1 and G2 / M ratios by 23.6% and 16.43%, respectively. Figure 4 (A to D). CCK-8 assay results for IGFBP3 cell lines showed that, compared with empty vector cells, the cell proliferation rate after IGFBP3 gene knockout increased by 8.61%, 12.69%, and 21.54% at 48h, 72h, and 96h, respectively (P<0.05). Figure 4 The above confirms that knocking out the IGFBP3 gene can promote cell proliferation.

[0120] Apoptosis in IGFBP3 knockout cells was analyzed using flow cytometry, and the results are as follows: Figure 5 As shown: the UU region represents necrotic cells, the UR region represents late-apoptotic cells, the LL region represents live cells, and the LR region represents early-apoptotic cells. Figure 5 (A to D), relative to the empty vector transfection group, IGFBP3 - / - The apoptosis rate decreased by 57.42% (P<0.05). Figure 5 (E).

[0121] In summary, this invention successfully obtained in vitro cultured IGFBP3. - / - The periosteum cell line of sika deer antler stalks (PPC) has been preliminarily verified that the IGFBP3 gene may exert a reverse regulatory role by inhibiting PPC cell proliferation and promoting apoptosis. The verification of the main function of the IGFBP3 gene provides more precise and effective data for molecular breeding of high-yielding sika deer and improving antler weight.

[0122] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of sgRNA targeting the deer IGFBP3 gene in promoting the proliferation of deer antler stem cells, characterized in that, The nucleotide sequence of the sgRNA is shown in at least one of SEQ ID NO: 9-12.

2. The application according to claim 1, characterized in that, The deer antler stem cells mentioned are deer antler regeneration stem cells.

3. The application according to claim 2, characterized in that, The deer antler regeneration stem cells were selected from horn stalk periosteum cells.

4. A method for increasing the weight of deer antlers, characterized in that, It includes: This results in the non-expression or suppressed expression of the IGFBP3 gene in deer.

5. The method for increasing the weight of deer antlers according to claim 4, characterized in that, Gene knockout was used to suppress or eliminate the expression of the IGFBP3 gene in deer.

6. The method for increasing the weight of deer antlers according to claim 5, characterized in that, The gene knockout is either a complete gene knockout or a conditional gene knockout.

7. The method for increasing the weight of deer antlers according to claim 6, characterized in that, The exon sequence of the IGFBP3 gene in deer was knocked out by gene knockout.

8. The method for increasing the weight of deer antlers according to claim 7, characterized in that, Knock out at least one exon sequence from the first and second exons of the IGFBP3 gene.

9. The method for increasing the weight of deer antlers according to claim 5, characterized in that, The gene knockout technique used is selected from at least one of the following: LEAPER technology, CRISPR / Cas gene editing technology, ZFN technology, TALEN technology, and Cre-loxp gene knockout technology; The CRISPR / Cas gene editing technology uses editing tools selected from any one of CRISPR / Cas9, CRISPR / Cas13, CRISPR / Cas12, and CRISPR / Cas14.

10. The method for increasing the weight of deer antlers according to claim 9, characterized in that, The gene knockout method includes infecting monoclonal deer antler stem cells with an sgRNA-Cas protein recombinant vector to achieve IGFBP3 gene knockout.

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