SgRNA molecule for targeted knockdown or knockout of IGFBP3 gene and application of sgRNA molecule in improvement of deer antler weight

Abstract

The invention discloses sgRNA molecules for targeted knockdown or knockout of IGFBP3 genes and application of the sgRNA molecules in improvement of deer antler weight, and relates to the technical field of antler regeneration. The IGFBP3 gene in the hornstalk periosteum cells of the sika deer is knocked out, so that proliferation and differentiation of antler stem cells can be promoted, cell apoptosis can be inhibited, and the cell apoptosis rate can be reduced. The proliferation of the pilose antler stem cells is promoted and the pilose antler weight is favorably improved. The verification of the IGFBP3 gene main body function provides more accurate and effective data for the molecular breeding of high-yield sika deer. Therefore, the inhibitor of the IGFBP3 gene and / or the inhibitor of the IGFBP3 protein is expected to improve the weight of the deer antler and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of antler regeneration, and in particular to a sgRNA molecule for targeted knockdown or knockout of an IGFBP3 gene and an application thereof in increasing antler weight. Background Art

[0002] Sika deer antler is a non-ossified, hairy young horn from male sika deer (Cervus nippon Temminck). It has been widely used in China for more than 2,000 years as a precious traditional animal medicine. Deer antler is the only mammalian organ that can regenerate rapidly every year without becoming cancerous, making it an ideal model for studying regeneration. 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 goal of sika deer farming.

[0003] The growth of antlers is mainly affected by genetics, nutritional conditions, and hormone levels. Insulin-like growth factor 1 (IGF-1) is a peptide growth factor that can promote 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 cell function through IGF-1-dependent mechanisms (involving IGF-1 signaling) and independent mechanisms (not involving IGF-1 signaling).

[0004] Currently, the associated genes that affect sika deer antler weight are still unclear.

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

[0006] The purpose of the present invention is to provide an application of an inhibitor of IGFBP3 gene and / or IGFBP3 protein in increasing the weight of velvet antler and a method for increasing the weight of velvet antler, thereby providing favorable technical support for increasing velvet antler yield.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a sgRNA molecule for targeted knockdown or knockout of the IGFBP3 gene, wherein the target sequence of the IGFBP3 gene targeted by the sgRNA is shown in at least one of SEQ ID NOs: 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 NOs: 9-12.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned sgRNA molecule for targeted knockdown or knockout of the IGFBP3 gene.

[0011] In a third aspect, the present invention provides a sgRNA molecule for targeted knockdown or knockout of the IGFBP3 gene or the use of the above-mentioned nucleic acid molecule in knocking down or knocking out the IGFBP3 gene in a cell.

[0012] In a fourth aspect, the present invention provides a method for knocking down or knocking out the IGFBP3 gene in a cell, comprising: knocking down or knocking out the IGFBP3 gene using the above-mentioned sgRNA molecule that targets the knockdown or knockout of 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 technology, CRISPR / Cas12 and CRISPR / Cas14.

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

[0015] In a fifth aspect, the present invention provides an application of an inhibitor of IGFBP3 gene and / or IGFBP3 protein in increasing the weight of deer antler.

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

[0017] In a seventh aspect, the present invention also provides a method for increasing the weight of deer antler, comprising: preventing or inhibiting the expression of the IGFBP3 gene of the deer.

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

[0019] The present invention annotates the candidate gene IGFBP3 associated with the sika deer antler weight through genome-wide association analysis (GWAS). Experiments show that the IGFBP3 gene plays an important regulatory role in the sika deer antler stalk periosteal cells (PPC). Knocking out the IGFBP3 gene in the sika deer antler stalk periosteal cells through CRISPR-Cas9 technology can promote the proliferation and differentiation of antler stem cells, inhibit cell apoptosis, and reduce the apoptosis rate. Promoting the proliferation of antler stem cells helps to increase antler weight. The verification of the main function of the IGFBP3 gene provides more accurate and effective data for the molecular breeding of high-yield sika deer. Therefore, inhibitors of the IGFBP3 gene and / or inhibitors of the IGFBP3 protein are expected to increase the antler weight, which is conducive to improving the accuracy and efficiency of the breeding of high-yield sika deer varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 The results of genome-wide association analysis of velvet weight trait. A: Manhattan plot of the significance of SNPs associated with velvet weight trait, the dotted line is the significant association threshold; B: QQ plot (quantile-quantile plot);

[0022] Figure 2 The figure is the restriction enzyme electrophoresis diagram (A) and sequencing comparison result diagram (B) of the recombinant plasmid after construction;

[0023] Figure 3 IGFBP3 - / - Results of gene knockout effect identification of cell lines (A: IGFBP3 - / - Cell sequencing results; B: Western blotting to detect the knockout effect of IGFBP3 gene; C: IGFBP3 - / - Expression of IGFBP3 protein in cells. “*” indicates WT VS IGFBP3 - / - , P<0.05, "#": Vector VS IGFBP3 - / - , P<0.05);

[0024] Figure 4 IGFBP3 - / - Effects of PI staining on PPC cell proliferation - / -Effects on PPC cell cycle; D: CCK8 method to detect IGFBP-3 - / - Effects on PPC cell proliferation;

[0025] Figure 5 IGFBP3 - / - Results of the experiment on the effect on apoptosis of PPC cells. A to D: Annexin V-PI double staining method to detect IGFBP3 - / - Effect on apoptosis of PPC cells; E: IGFBP3 - / - Statistics of the total apoptosis rate of PPC cells. “*” indicates WTVS IGFBP3 - / - , P<0.05, "#": Vector VS IGFBP3 - / - , P<0.05). DETAILED DESCRIPTION

[0026] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0027] Antler weight of sika deer is the most important genetic trait in molecular breeding. Therefore, clarifying the function of key candidate genes that regulate antler weight traits will help improve the accuracy and efficiency of high-yield sika deer breeding. In particular, by knocking out the IGFBP3 gene in the periosteal cells of the sika deer antler stalk, it was found that IGFBP3 inhibits the proliferation and promotes apoptosis of antler stem cells. The determination of the main function of the IGFBP3 gene provides more accurate and effective data for the molecular breeding of high-yield sika deer. Inhibitors of the IGFBP3 gene are expected to increase antler weight, which will help improve the accuracy and efficiency of high-yield sika deer breeding.

[0028] In a first aspect, the present invention provides a sgRNA molecule for targeted knockdown or knockout of the IGFBP3 gene, wherein the target sequence (or targeting sequence) of the IGFBP3 gene targeted by the sgRNA is shown in at least one of SEQ ID NOs: 1-8.

[0029] Table 1. Targeting 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 NOs: 9-12:

[0032] SEQ ID NO:9:

[0033] CGGCUCGUGCCGCAGGCGG. Against targeting sequence 1.

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

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

[0036] SEQ ID NO: 12: AUGCUUGGUCUUCUUCCG. Targeting sequence 4.

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

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

[0039] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned sgRNA molecule for targeted knockdown or knockout of the IGFBP3 gene.

[0040] In a third aspect, the present invention provides the use of a sgRNA molecule that targets and knocks down or knocks out the IGFBP3 gene in knocking down or knocking out the IGFBP3 gene in a cell.

[0041] In a fourth aspect, the present invention provides a method for knocking down or knocking out the IGFBP3 gene in a cell, comprising: knocking down or knocking out the IGFBP3 gene using the above-mentioned sgRNA molecule that targets the knockdown or knockout of 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 technology, CRISPR / Cas12 and CRISPR / Cas14.

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

[0044] In a fifth aspect, the present invention provides an application of an inhibitor of IGFBP3 gene and / or IGFBP3 protein in increasing the weight of deer antler.

[0045] The above-mentioned increase in antler weight includes but is not limited to: accelerating the growth and development of antler tissue, increasing the weight of antler, and shortening the antler production cycle.

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

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

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

[0049] In a preferred embodiment of the present invention, antler regeneration stem cells are selected from horn stalk periosteal cells. Antler horn stalk periosteal cells belong to mesenchymal stem cells. The formation of cartilage is driven by the proliferation and differentiation of mesenchymal stem cells into chondrocytes. The experimental results of the present invention show that the IGFBP3 gene is an inhibitor of cartilage differentiation, and the apoptosis of antler stem cells caused by the IGFBP3 gene may eventually inhibit the formation of antler cartilage. Thereby increasing the weight of antler velvet.

[0050] In a preferred embodiment of the present invention, the substance inhibits the IGFBP3 gene level and / or IGFBP3 protein level or activity in the cells of the sensitized zone of the horn shank 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 modified or unmodified molecules:

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

[0053] In a preferred embodiment of the present invention, the modification is chemical modification, and the chemical modification is selected from one or more of methoxy modification, fluorine modification, phosphorothioate modification, LNA modification and methoxyethyl modification. The 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 comprises an sgRNA expression vector and an expression vector of a Cas protein, and 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 targeting the first exon and the second exon of the IGFBP3 gene;

[0056] In a preferred embodiment of the present invention, the sequence targeted by the sgRNA is shown in at least one of SEQ ID NOs: 1-8.

[0057]

[0058] In a sixth aspect, the present invention also provides an application of an inhibitor of the IGFBP3 gene in the proliferation of antler stem cells. The inventors have found that inhibiting the expression of the IGFBP3 gene can help reduce the number of cells in the G0 / G1 and G2 / M phases, promote the proliferation of antler stem cells, and thus increase the weight of antler velvet.

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

[0060] In a preferred embodiment of the present invention, the antler regeneration stem cells are selected from horn stalk periosteal 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 modified or unmodified molecules:

[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 antler, comprising: preventing or inhibiting the expression of the IGFBP3 gene of the deer.

[0064] In a preferred embodiment of the present invention, the IGFBP3 gene of deer is not expressed or its expression is inhibited by gene knockout;

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

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

[0067] In a preferred embodiment of the present invention, at least one exon sequence of the first exon and the second exon 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: LEAPER technology, CRISPR / Cas gene editing technology, ZFN technology, TALEN technology and Cre-loxp gene knockout technology.

[0069] LEAPER technology is a technology that relies on introducing a segment of RNA (arRNA) that is complementary to the sequence of the target site into the cell to recruit ADAR protein to edit the RNA target site. The RNA used in this technology that is complementary to the sequence of the target site can stimulate more 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 technology, CRISPR / Cas12 and CRISPR / Cas14;

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

[0072] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0073] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0074] Material:

[0075] Pedicle periosteal cells (PPCs) and 293T cells were provided by the antler stem cell team of the Institute of Specialty Products, Chinese Academy of Agricultural Sciences. Lenti-Guide-Puro plasmid was purchased from Changsha Fenghui Biological Co., Ltd. Stbl3 competent cells and Trans1-T1 competent cells were purchased from Beijing Quanshijin Biological Co., Ltd. Cells were cultured with DMEM medium (Gibco, C11995500BT), FBS (Sigma-Aldrich, F8318), trypsin (Gibco, 25200056), double antibody (Gibco, 15140-122), cell freezing solution (Beyotime, C0210), and PBS (Beyotime, C0221A). Plasmids were extracted using Beijing Tiangen Technology Plasmid Mini-Extraction Kit (DP103), Endotoxin-Free Plasmid Max-Extraction Kit (DP117), and Common Agarose Gel DNA Recovery Kit.

[0076] Example 1

[0077] This example provides an experimental method for the GWAS annotation of the heavy association between IGFBP3 and sika deer antler.

[0078] Genome-wide association analysis methods:

[0079] During the 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 effects 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 is the phenotypic trait, X is the indicator matrix of fixed effects, α is the estimated parameter of fixed effects; Z is the indicator matrix of SNPs, β is the effect of SNPs; W is the indicator matrix of random effects, μ is the predicted random individual, e is the random residual, and obeys e~(0,δe2).

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

[0083] result:

[0084] The GEMMA software was used to perform genome-wide association analysis on velvet weight traits, and the Manhattan plot was obtained ( Figure 1 A in Figure ) and QQ in Figure . Figure 1The actual P value in the QQ plot deviates slightly from the null hypothesis P value of no association only in the tail region, indicating that the trait difference is not caused by population stratification. The GWAS results show that 9 SNPs reached a significant level (p < 1e-6), see Figure 1 The IGFBP3 gene was present at 22.8 kb from the g.27759264A>G site, see Table 2 .

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

[0086]

[0087] Example 2

[0088] This example constructs and identifies a recombinant plasmid.

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

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

[0091] Table 1. Targeting sequences

[0092]

[0093] Table 2. sgRNA sequences

[0094]

[0095] At 4°C, the Lenti-Guide-Puro plasmid was digested with BsmBI endonuclease, and the digested plasmid was recovered by electrophoresis and gel cutting ( Figure 2 A in the figure). Dilute the annealed DNA coding sequence of the sgRNA shown in Table 2 200 times on an ice box to obtain an insert fragment solution. Prepare the reaction system on an ice box according to Table 2, mix by pipetting, centrifuge, and place at room temperature for 15 minutes. Then transform it into Stbl3 competent cells, spread it on LB solid culture and culture it at 37°C overnight. Pick a single clone colony and send it to Shanghai Sangon Biotech Co., Ltd. for sequencing verification. The recombinant vector that has been successfully sequenced and verified is extracted for plasmid and placed at -80°C for standby use. The sequencing results show that the DNA coding fragments of the four sgRNAs are all inserted into the corresponding positions of the vector, and the recombinant plasmid is successfully constructed ( Figure 2 (middle B).

[0096] Table 2. sgRNA DNA coding sequence and linear vector connection system

[0097]

[0098] Example 3

[0099] Identification of the effect of gene knockout of IGFBP3 cell lines.

[0100] The experimental method is as follows:

[0101] 1. Cell Infection

[0102] The lentivirus was placed at 4°C for melting and used, and the transfection complex was prepared according to the following groups: g1 / 2 / 3 / 4 recombinant vector group (5μg / mL polybrene medium + recombinant vector virus), g0 empty vector group (5μg / mL polybrene medium + empty vector), blank group (5μg / mL polybrene medium). After returning to the incubator for 12 hours, replace with completely fresh medium and continue culturing. After 72 hours of infection, the original medium was discarded and replaced with a complete medium containing 5μg / mL blasticidin and 4μg / mL puromycin. The infected cells were screened for double drugs. The blank group was used as a control. After the blank group died completely, the recombinant vector group and the empty vector group continued to be cultured for 96 hours. When the cell fusion reached more than 85%, the cells were passaged.

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

[0104] After the double-drug screening, the cells were digested and counted with trypsin, and then inoculated in a six-well plate at a density of 1000 cells / well to keep the cells suspended. Under a microscope, a single suspended cell was aspirated with a micropipette, inoculated in a 96-well plate, and 100 μL of complete culture medium containing 20% ​​FBS was added, and returned to the 37°C, 5% CO2 cell culture incubator to continue to expand the culture and obtain a monoclonal cell line. When the cells grow into colonies, they are digested with trypsin and transferred to a 24-well plate to continue to expand the culture. The DNA of wild-type PPC and lentivirus-infected PPC was extracted according to the instructions of the DNA extraction kit and stored at -20°C. PCR amplification of DNA fragments: Using wild-type and monoclonal cell line DNA as templates, primers are used to use high-fidelity DNA polymerase to amplify DNA fragments containing the Cas9 protein cleavage target. Recover the DNA target fragment, measure the concentration with an ultraviolet spectrophotometer, and gently mix the DNA fragment with the pEASY cloning vector according to the kit instructions in the correct proportion. The ligated product is transformed into the Trans-T1 competent state according to the transformation steps; take IPTG and X-gal and spread them evenly on LB solid medium (containing AMP), and place them in a 37℃ oven for 30 minutes to promote the absorption of IPTG and X-gal. Then take 50μ of bacterial liquid and spread them evenly on LB solid medium, culture at 37℃ for 12 hours, pick out white single clones, inoculate them in 5ml LB liquid medium (containing AMP), culture at 180r / min, 37℃ for 12 hours, and send them to Shanghai Biotech for sequencing.

[0105] 3. Detection of IGFBP3 protein expression by Western blotting

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

[0107] According to the above method, the recombinant plasmid was co-transfected into PPC for monoclonal cell culture. After DNA was extracted from the monoclonal cells, the target fragment was amplified by PCR and connected to the pEASY vector. Sequencing showed that the survival rate of single cells infected with G3 lentivirus was also the highest, and there were three types of mutations. Western-blot was used to detect the expression of IGFBP3 protein in the three mutant types of cells. The results showed that the No. 2 mutant IGFBP3 protein was completely knocked out, proving that the IGFBP3 monoclonal cell line with IGFBP3 gene knockout was successfully constructed ( Figure 3A and B in Figure 2). After comparing the sequencing results of mutant 2 with the complete gene sequence of IGFBP3, it was found that the monoclonal cell line was missing 83 bases in the IGFBP3 allele ( Figure 3 Middle 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 to detect cell cycle

[0112] Wild-type cells, empty vector-transfected cells, IGFBP3 - / - When the cells were cultured to 85%, the cells were collected and centrifuged at 1000 r / min for 5 min to obtain the cell pellet, which was fixed with 70% ethanol at 4°C for 24 h. After fixation, 500 μL of propidium iodide staining solution was added to each tube of cell sample, and the cell pellet was slowly and fully resuspended, and incubated at 37°C in the dark for 30 min. The flow cytometer detected red fluorescence at an excitation wavelength of 488 nm and light scattering at the same time.

[0113] 2. CCK8 method to detect cell proliferation rate

[0114] Wild-type cells, empty vector-transfected cells, IGFBP3 - / - The cells were cultured to the logarithmic phase and then subcultured to a 96-well plate. The cells were counted by a cell counter, and 2,000 cells were inoculated in each well. The cells were cultured for 24 hours at 37°C and 5% CO2. The complete culture medium (10% FBS) containing 10% CCK8 reagent was replaced, and the cells were cultured for another 3 hours in a cell culture incubator. The absorbance was measured at a wavelength of 450 nm using an ELISA reader. This operation was performed every 24 hours, and the absorbance of three groups of cells was obtained at 24 hours, 48 ​​hours, 72 hours, and 96 hours. The data were statistically analyzed.

[0115] 3. Annexin V-PI double staining to detect cell apoptosis

[0116] Wild-type cells, empty vector-transfected cells, IGFBP3 - / - When the cells were cultured to 85%, the cells were collected and centrifuged at 1000r / min for 5min. The cell pellet was resuspended in 500μL 1xBinding Buffer. 5μL Annexun V-FITC and 5μL Propidium lodide were added, gently mixed, and incubated at room temperature in the dark for 20min. A negative control group (i.e., normal cells cultured without Annexun V-FITC and Propidium lodide) was set up at the same time. The cells were tested by flow cytometry.

[0117] 4. Statistical Analysis

[0118] Excel was used for data entry and SPSS22.0 statistical software was used for data analysis. The data were expressed as mean ± standard deviation. One-way analysis of variance was used for multiple group comparisons. P < 0.05 indicated statistically significant differences. GraphPad Prism 8.0 was used to draw bar graphs.

[0119] The results of cell cycle detection showed that compared with empty vector cells, G0 / G1 and G2 / M decreased by 23.6% and 16.43% respectively after IGFBP3 gene knockout. Figure 4 A to D in the middle). The results of CCK-8 detection of 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 (E), the above confirmed that knocking out the IGFBP3 gene can promote cell proliferation.

[0120] Flow cytometry was used to analyze the apoptosis of cells after IGFBP3 knockout. Figure 5 As shown: UU area is the necrotic area, UR area is the late apoptotic cells, LL area is the living cells, and LR area is the early apoptotic cells ( Figure 5 (A to D), compared with the empty vector transfection group, IGFBP3 - / - The apoptosis rate decreased by 57.42% (P<0.05) ( Figure 5 Middle E).

[0121] In summary, the present invention successfully obtained IGFBP3 cultured in vitro - / - The sika deer antler pedunculate periosteal cell line (PPC) preliminarily verified that the IGFBP3 gene may play a reverse regulatory role by inhibiting PPC cell proliferation and promoting cell apoptosis. The verification of the main function of the IGFBP3 gene provides more accurate and effective data for the molecular breeding of high-yield sika deer and increasing the weight of antlers.

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An sgRNA molecule for targeted knockdown or knockout of the IGFBP3 gene, characterized in that: The target sequence of the IGFBP3 gene targeted by the sgRNA is shown in at least one of SEQ ID NOs: 1-8.

2. The sgRNA molecule for targeted knockdown or knockout of the IGFBP3 gene according to claim 1, characterized in that: The crRNA nucleotide sequence of the sgRNA molecule is shown in at least one of SEQ ID NOs: 9-12.

3. A nucleic acid molecule, characterized in that It encodes the sgRNA molecule for targeted knockdown or knockout of the IGFBP3 gene as described in any one of claims 1-2.

4. Use of the sgRNA molecule for targeted knockdown or knockout of the IGFBP3 gene according to any one of claims 1 to 2 or the nucleic acid molecule according to claim 3 in knocking down or knocking out the IGFBP3 gene in a cell.

5. A method for knocking down or knocking out the IGFBP3 gene in a cell, characterized in that: The method comprises: using the sgRNA molecule for knocking down or knocking out the IGFBP3 gene as described in any one of claims 1 to 2 or the nucleic acid molecule as described in claim 3 to knock down or knock out the IGFBP3 gene; Preferably, when knocking down or knocking out the IGFBP3 gene, the gene editing tool used is selected from any one of CRISPR / Cas9, CRISPR / Cas13 technology, CRISPR / Cas12 and CRISPR / Cas14.

6. The method for knocking down or knocking out the IGFBP3 gene in a cell according to claim 5, characterized in that: The method of knocking down or knocking out the IGFBP3 gene includes: infecting the monoclonal antler stem cells with the sgRNA-Cas protein recombinant vector to achieve the knockout of the IGFBP3 gene.

7. Application of IGFBP3 gene inhibitors and / or IGFBP3 protein inhibitors in the proliferation of antler stem cells; Preferably, the antler stem cells are antler regeneration stem cells; Preferably, the antler regeneration stem cells are selected from horn stalk periosteal cells.

8. The use according to claim 7, characterized in that: The inhibitor of the IGFBP3 gene is selected from at least one of the following molecules, modified or unmodified: siRNA, sgRNA and Cas proteins, snoRNA, shRNA and microRNA; Preferably, the nucleotide sequence of the sgRNA is shown in at least one of SEQ ID NOs: 9-12.

9. A method for increasing the weight of velvet antler, characterized in that: It includes: This results in the deer IGFBP3 gene not being expressed or having its expression inhibited.

10. The method for increasing the weight of velvet antler according to claim 9, characterized in that: By knocking out the gene, the IGFBP3 gene of deer is not expressed or its expression is inhibited; Preferably, the gene knockout is a complete gene knockout or a conditional gene knockout; Preferably, the exon sequence of the deer IGFBP3 gene is knocked out by gene knockout; Preferably, at least one exon sequence of the first exon and the second exon of the IGFBP3 gene is knocked out; Preferably, the gene knockout adopts a gene editing technology selected from at least one of LEAPER technology, CRISPR / Cas gene editing technology, ZFN technology, TALEN technology and Cre-loxp gene knockout technology; Preferably, the editing tool used in the CRISPR / Cas gene editing technology is selected from any one of CRISPR / Cas9, CRISPR / Cas13 technology, CRISPR / Cas12 and CRISPR / Cas14; Preferably, the gene knockout method comprises: infecting monoclonal antler stem cells with a sgRNA-Cas protein recombinant vector to achieve knockout of the IGFBP3 gene.

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