Method for establishing a mouse model of conditional knockout of OGT gene in embryonic limb bud and application thereof

Abstract

The application discloses a kind of establishment and its application of OGT gene conditional knockout mouse model in embryo limb bud.The Cas9 mRNA and gRNA are obtained by in vitro transcription method;Homologous recombination vector is constructed by the method of In-Fusion cloning.Cas9 mRNA, gRNA and donor vector are microinjected into fertilized egg of C57BL / 6J mouse, and F0 generation mouse is obtained.The application finds that there is obvious skeletal deformity after conditional knockout OGT in embryo limb bud for the first time;For subsequent OGT gene in the function research of bone development and bone formation provides good animal model;It can be directly used for the mechanism research and drug research / efficacy evaluation of osteoarthritis to provide effective experimental animal model, with important application value.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to the establishment and application of a conditional knockout mouse model of the OGT gene in embryonic limb buds. Background Technology

[0002] O-GlcNAc glycosylation is an important post-translational modification of proteins that links N-acetylglucosamine to the hydroxyl groups of serine or threonine residues in a target protein. It is widely involved in almost all vital activities, including gene transcription, cell cycle progression, signal transduction, and maintenance of metabolic homeostasis. Imbalances in this modification system are closely related to the pathological processes of many major diseases.

[0003] O-GlcNAc transferase (OGT) is the only key enzyme catalyzing this modification process. The OGT gene is located on the X chromosome. Patients with OGT mutations, in addition to neurodevelopmental abnormalities, may exhibit phenotypes such as finger (toe) deformities and short stature. Numerous studies have demonstrated that OGT plays a crucial regulatory role in bone metabolism (such as osteogenic differentiation and osteoporosis). In age-related osteoarthritis, OGT maintains chondrocyte homeostasis and prevents cartilage degeneration by stabilizing FoxO1 protein. For example, OGT expression is downregulated in patients with osteoporosis. Functional studies of OGT in the skeletal system have revealed its potential as a therapeutic target for bone diseases. However, research on the role of OGT in bone development and bone diseases remains limited.

[0004] Therefore, it is necessary to construct an OGT gene knockout model to provide an effective experimental animal model for studying the role of OGT in bone development, the mechanism of osteoarthritis, and drug development / efficacy evaluation. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide a conditional knockout mouse model of the OGT gene in embryonic limb buds and its application. The present invention utilizes CRISPR / Cas9 technology to construct a mouse model in which OGT is knocked out in embryonic limb buds.

[0006] The technical solution of the present invention is as follows:

[0007] A first aspect of the present invention provides a method for establishing a conditional knockout mouse model of the OGT gene in embryonic limb buds, comprising the following steps: (1) The specific target site of the OGT gene was determined, and Cas9 mRNA and gRNA were obtained by in vitro transcription; the gRNA includes gRNA1 and gRNA2, wherein the nucleic acid sequences of gRNA1 and gRNA2 are SEQ ID NO: 1 and SEQ ID NO: 2, respectively; (2) A homologous recombination donor vector was constructed by in-fusion cloning; the donor vector includes a 5' homologous arm, a flux region and a 3' homologous arm; the nucleic acid sequence of the donor vector is SEQ ID NO: 3; (3) Cas9 mRNA, Donor vector and gRNA were microinjected into fertilized eggs of donor mice, and the fertilized eggs were then transplanted into recipient rats to obtain OGT. flox / + Mice; (4) Select OGT flox / + Mice were self-crossed and propagated to obtain homozygous OGT. flox / flox Mice, selected OGT flox / flox Female mouse for later use; (5) Prx1 cre transgenic mice were mated with C57BL / 6J mice to obtain Prx1 cre transgenic male mice; (6) Obtain the OGT flox / flox Female mice were mated with the obtained Prx1 cre transgenic male mice. Because OGT is located on the X chromosome, the offspring that were Cre-positive were the conditionally knocked-out OGT mouse model, i.e., OGT. flox / y -Prx1cre mice.

[0008] The present invention also provides the above-described method for identifying the genotype of mice.

[0009] Preferably, the method for identifying the mouse genotype includes a PCR method, and the primers used in the PCR method include OGT. flox Forward primer OGT flox F, OGT flox Reverse primer OGT flox R, Prx1 cre forward primer Prx1 creF, Prx1 cre reverse primer Prx1 creR; the OGT flox The nucleic acid sequence of F is SEQ ID NO: 4, OGT flox The nucleic acid sequence of R is SEQ ID NO: 5; the nucleic acid sequence of Prx1 creF is SEQ ID NO: 6; and the nucleic acid sequence of Prx1 creR is SEQ ID NO: 7.

[0010] A second aspect of the present invention provides the application of an animal model constructed by the method described in the first aspect of the present invention in any of the following: (1) Screening for drugs for osteoarthritis; (2) Screening for drugs for osteoporosis; (3) Functional study of OGT gene in bone development and bone formation.

[0011] A third aspect of the present invention provides a kit comprising the Cas9 protein, gRNA, donor vector, and primers as shown in SEQ ID NO:4 to SEQ ID NO:7 as described in the first aspect of the present invention.

[0012] The beneficial effects of this invention are as follows: This invention provides a method for establishing a mouse model of conditional knockout of the OGT gene in embryonic limb buds; this invention is the first to discover that conditional knockout of OGT in embryonic limb buds results in significant skeletal deformities; it provides an excellent animal model for subsequent functional studies of the OGT gene in skeletal development and bone formation; it can be directly used for mechanism studies of osteoarthritis and drug development / efficacy evaluation, providing an effective experimental animal model with significant application value.

[0013] This invention demonstrates that the protein-coding gene OGT and its upstream and downstream biological pathways can be used as novel drug targets for the treatment of osteoarthritis. Attached Figure Description

[0014] Figure 1 This invention, OGT flox / y -Schematic diagram of the Prx1 cre mouse construction strategy; Figure 2 The OGT obtained in Embodiment 1 of this invention flox / y Photograph of PCR genotyping results for Prx1 cre mice; Figure 3 This is a photograph of a mouse with conditional OGT gene knockout obtained in Example 1 of this invention, at day P0 after birth; Figure 4 These are Western blot images of limb bone tissue from mice with conditionally knocked-out OGT gene at day P0 after birth, obtained in Example 1 of this invention. Figure 5 This is a photograph of the skeleton of a mouse with conditionally knocked-out OGT gene one week after birth, showing significant differences in bone formation after staining with Alcian blue and Alizarin Red. This is Example 2 of the present invention. Detailed Implementation

[0015] The present invention will be further illustrated below with reference to specific embodiments. These examples are merely illustrative and not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0016] This invention utilizes the principle of homologous recombination, employing homologous recombination in fertilized eggs to modify the OGT gene via flux. The simplified process is as follows: Cas9 mRNA and gRNA are obtained through in vitro transcription; a homologous recombination vector (donor vector) containing a 5' homologous arm, the flux region between exons 2 and 3 of the FKBP10 gene, and a 3' homologous arm is constructed using in-fusion cloning. The Cas9 mRNA, gRNA, and donor vector are then microinjected into fertilized eggs of C57BL / 6J mice to obtain F0 generation mice. A schematic diagram of the construction strategy is shown below. Figure 1 As shown.

[0017] Example 1: Construction of a conditional knockout mouse model of OGT gene in embryonic limb buds 1.1OGT flox / + Feeding of mice and Prx1 Cre transgenic mice: OGT flox / + The mice and Prx1 Cre transgenic mice were fed in accordance with SPF animal feeding standards. After isolation and observation without any abnormalities, they were put into the feeding area and experimental operations were carried out in strict accordance with the relevant SPF animal management regulations.

[0018] 1.2 OGT flox / + Mouse genotyping OGT was extracted from the tail of a mouse. flox / + Mouse genomic DNA was used for mouse genotyping with primers; the specific primer sequence was designed as follows: OGT flox The nucleic acid sequence of F is SEQ ID NO: 4, OGT flox The nucleic acid sequence of R is SEQ ID NO: 5.

[0019] Figure 2 PCR identification results showed two bands amplified: 186bp and 253bp. The 186bp band indicated that the OGT gene was not marked by the Loxp site, while the 253bp band indicated that the OGT gene was marked by the Loxp site. The simultaneous appearance of both bands indicates OGT. flox / + OGT has only one 253bp stripe. flox / flox .

[0020] 1.3 Genotyping of Prx1Cre transgenic mice Mouse tails were cut off, and genomic DNA of Prx1Cre transgenic mice was extracted. Genotyping of the mice was performed by PCR using a pair of primers. The specific design sequences are as follows: the nucleic acid sequence of Prx1 creF is SEQ ID NO: 6, and the nucleic acid sequence of Prx1 creR is SEQ ID NO: 7.

[0021] Figure 2 The PCR identification results showed that 217 bp bands were obtained, indicating that the Cre gene exists on a homologous chromosome of the embryonic limb bud marker gene prx1. The absence of bands indicates that the Cre gene does not exist on the homologous chromosome of the embryonic limb bud marker gene prx1, thus proving that the obtained mouse is the target Prx1 Cre transgenic mouse.

[0022] 1.4 Establishment of a mouse model of conditional knockout of OGT gene in embryonic limb buds (1) The specific target site of the OGT gene was determined, and Cas9 mRNA and gRNA were obtained by in vitro transcription. The gRNA included gRNA1 and gRNA2, and the nucleic acid sequences of gRNA1 and gRNA2 were SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The homologous recombination donor vector was constructed by in-fusion cloning. The donor vector included a 5' homologous arm, a flux region and a 3' homologous arm. The nucleic acid sequence of the donor vector was SEQ ID NO: 3. Cas9 mRNA, Donor vector and gRNA were microinjected into the fertilized eggs of donor mice, and the fertilized eggs were then transplanted into recipient rats to obtain OGT. flox / + Mice. The obtained OGT flox / + Mice were self-crossed and propagated to obtain homozygous OGT. flox / flox Mice, selected OGT flox / flox Female mouse for later use; (2) Prx1 cre transgenic mice were mated with SPF-grade C57BL / 6J mice to obtain Prx1 cre transgenic male mice; (3) Obtain the OGT flox / flox Female mice were mated with the obtained Prx1 cre transgenic male mice. Because OGT is located on the X chromosome, the offspring that were Cre-positive were the conditionally knocked-out OGT mouse model, i.e., OGT. flox / y -Prx1cre mice.

[0023] (4) The obtained OGT flox / flox Mice, Prx1 cre mice, OGT flox / + -Prx1 cre mice and OGTflox / y -Prx1 cre mice were genotyped using PCR with specific primers. Primers included OGT. flox Forward primer OGT flox F, OGT flox Reverse primer OGT flox R, Prx1cre forward primer Prx1creF, Prx1cre reverse primer Prx1creR; the OGT flox The nucleic acid sequence of F is SEQ ID NO: 4, OGT flox The nucleic acid sequence of R is SEQ ID NO: 5; the nucleic acid sequence of Prx1creF is SEQ ID NO: 6; and the nucleic acid sequence of Prx1creR is SEQ ID NO: 7.

[0024] Prx1 Cre male mouse with OGT flox / flox Female mice mate to obtain more OGT. flox / y -Prx1 cre mice (male mice in the experimental group) and OGT flox / y Mice (male control group). Genomic DNA was extracted from the tails of offspring mice around days P0 and P7. Genotypes of the offspring mice were identified by PCR using primers OGT and Prx1cre. Once the offspring genotypes were determined, they were used for phenotypic identification of a conditional knockout OGT gene mouse model in embryonic limb buds.

[0025] Figure 3 The image shows a comparison of mice in the experimental and control groups at day P0 after birth. As shown in the figure, the experimental group mice exhibited significantly underdeveloped limbs compared to their littermate control counterparts.

[0026] 1.5 Changes in bone tissue protein expression in conditionally knocked-out OGT gene mice (1) Extraction of protein from mouse bone tissue: Collect the limbs of mice, remove the skin and flesh, carefully remove the connective tissue under a stereomicroscope, leaving only the long bones of the limbs; use a bone tissue protein extraction kit to prepare the extraction solution on ice according to the ratio; rapidly freeze and grind the bone tissue with liquid nitrogen, and add 150 μl of bone tissue extraction solution to each sample. Place the sample on ice for 45 min to lyse, and then centrifuge at 12000 rpm for 12 min at 4℃; then aspirate the supernatant (protein solution) into a new EP tube and place it on ice; after the protein concentration is determined, add Loading Buffer and β-mercaptoethanol in a ratio of supernatant: Loading Buffer: β-mercaptoethanol = 60:20:1; place in a 95℃ metal bath for 5 min; then the protein sample can be stored in a -80℃ freezer.

[0027] (2) Determination of protein concentration in mouse bone tissue: BCA working solution was prepared using the BCA protein assay kit, with a ratio of A to B of 50:1. 200 μl of working solution was required for each sample. After mixing, the solution was added to each well of a 96-well plate. According to the standard curve, the sample was diluted 5 times with ddH2O, mixed, and 9 μl of the solution was added to each well of the 96-well plate. The 96-well plate was gently shaken and incubated at 37°C for 30 min. The absorbance of the protein sample was measured using an ELISA reader. Based on the measured standard curve values, a protein concentration-absorbance standard curve was plotted. The protein concentration and loading amount of the sample were calculated based on the sample absorbance.

[0028] (3) Western blot experiment: First, prepare an SDS-PAGE stacking gel and a 10% separating gel. Install the prepared gels in the electrophoresis tank and add 800 ml of 1X Running Buffer. Add 5 μl of Protein Maker and the same total protein amount of sample to each well. Use 80V; after the protein passes through the upper stacking gel, adjust the voltage to 120V. Preparation before transfer: Prepare 1X Trans Buffer and pre-cool it at 4°C. Activate the cut PVDF membrane by immersing it in methanol for 30 seconds. Pour the pre-cooled 1X Trans Buffer into a container and immerse it in a clip with filter paper and sponge. Cut the separating gel from the glass plate and place it on the filter paper, protein side up. Lay the activated PVDF membrane flat on top of the separating gel, place it in the transfer tank, add 1X Trans Buffer and ice, cover, and place the entire transfer tank in a foam box filled with ice. Turn on the power; the transfer condition is 200 mA. 2 hours; Immerse in blocking buffer (5% skim milk) at room temperature on a shaker (50 rpm) for 1 hour; then discard the blocking buffer, pour in 1X PBST and wash on a shaker (160 rpm) for 5 minutes, then cut according to the size of the target protein; Incubate primary antibody: Prepare primary antibody: Dilute the antibody stock solution 1000 times and add it to the antibody dilution buffer (5% BSA); dilute the internal control GAPDH 3000 times; place the PVDF membrane corresponding to the band size of the target protein into the antibody dilution buffer for the target protein, and incubate overnight on a shaker at 4°C; Incubate secondary antibody: Recover the primary antibody, pour in 1X PBST and wash on a shaker (160 rpm) for 10 minutes × 3 times; then add the corresponding secondary antibody, which is added to the antibody dilution buffer (5% BSA) at a ratio of 1:3000 and incubate on a shaker at room temperature for 1 hour; then place in 1X PBST on a shaker (160 rpm) for 1 hour. Wash for 10 min × 3 times (rpm); Observation results: Millipore developer was added to the PVDF membrane using a Biorad gel imaging system for development, and the images were saved for subsequent quantitative analysis of gray values ​​using Imagej software.

[0029] like Figure 4 As shown, OGT was knocked out in the bone tissue of the limbs, indicating that the constructed knockout animal model is effective.

[0030] Example 2 Identification of the phenotype of conditionally knocked-out OGT gene mice in embryonic limb buds 2.1 Effects of conditional knockout of the OGT gene on skeletal development in newborn mice Select OGT 7 days after birth flox / y -Prx1cre male mice and OGT flox / y Male mice were dissected to obtain limb bones, which were then stained with Alizarin Red-Alixine Blue. The specific steps are as follows: 2.1.1 Mice were euthanized after being anesthetized for 7 days with 5% sodium pentobarbital.

[0031] 2.1.2 Carefully remove the soft tissue from the limbs of the suckling mouse as follows: The mouse's limbs were cut off and placed in 65-degree water for 10 minutes. Under a stereomicroscope, the skin, muscles, connective tissue, etc. of the limbs were carefully dissected.

[0032] 2.1.3 Fix the sample with 95% alcohol for 24 hours.

[0033] 2.1.4 Transfer the sample to acetone and soak it overnight (10-12 h) at room temperature to remove the fat from the sample.

[0034] 2.1.5 After rinsing the sample three times with deionized water, immerse it completely in 0.03% Alcian blue for 24 hours to stain the cartilage. Due to seasonal temperature differences, the specific staining time can be adjusted according to the degree of staining of the cartilage.

[0035] 2.1.6 Change the solution twice with 75% alcohol, then incubate overnight at room temperature with 95% alcohol.

[0036] 2.1.7 Soak in 1% KOH at room temperature for 1 hour and 10 minutes, then soak completely in Alizarin Red for 3 hours and 20 minutes to stain the osteogenic components of the sample.

[0037] 2.1.8 After staining, place the sample in 50% glycerol + 50% 1% KOH at room temperature to slowly remove excess fat until the color is clear, and finally store in 100% glycerol.

[0038] 2.1.9 Take photos to record, observe and analyze.

[0039] from Figure 5 It can be observed that, compared with the control group, OGT flox / y Compared to suckling mice, the control group OGT flox / y-Prx1 cre suckling mice have obvious maldevelopment of the upper limbs, showing obvious symptoms of skeletal development defects.

Claims

1. A method for establishing a conditional knockout mouse model of the OGT gene in embryonic limb buds, characterized in that, The establishment method includes the following steps: (1) The specific target site of the OGT gene was determined, and Cas9 mRNA and gRNA were obtained by in vitro transcription; the gRNA includes gRNA1 and gRNA2, wherein the nucleic acid sequences of gRNA1 and gRNA2 are SEQ ID NO: 1 and SEQ ID NO: 2, respectively; (2) A homologous recombination donor vector was constructed by in-fusion cloning; the donor vector includes a 5' homologous arm, a flux region and a 3' homologous arm; the nucleic acid sequence of the donor vector is SEQ ID NO: 3; (3) Cas9 mRNA, Donor vector and gRNA were microinjected into fertilized eggs of donor mice, and the fertilized eggs were then transplanted into recipient rats to obtain OGT. flox / + Mice; (4) Select OGT flox / + Mice were self-crossed and propagated to obtain homozygous OGT. flox / flox Mice, selected OGT flox / flox Female mouse for later use; (5) Prx1 cre transgenic mice were mated with C57BL / 6J mice to obtain Prx1 cre transgenic male mice; (6) Obtain the OGT flox / flox Female mice were mated with the obtained Prx1 cre transgenic male mice. Because OGT is located on the X chromosome, the offspring that were Cre-positive were the conditionally knocked-out OGT mouse model, i.e., OGT. flox / y -Prx1cre mice.

2. The method for establishing a transgenic animal model as described in claim 1, characterized in that, The method also includes identifying the genotype of the mouse.

3. The method for establishing a transgenic animal model according to claim 2, characterized in that, The methods for identifying the mouse genotype include PCR, and the primers used in the PCR method include OGT. flox Forward primer OGT flox F, OGT flox Reverse primer OGT flox R, Prx1 cre forward primer Prx1 creF and Prx1 cre reverse primer Prx1 creR; the OGT flox The nucleic acid sequence of F is SEQ ID NO: 4, OGT flox The nucleic acid sequence of R is SEQ ID NO: 5; the nucleic acid sequence of Prx1 creF is SEQ ID NO: 6; and the nucleic acid sequence of Prx1 creR is SEQ ID NO:

7.

4. The conditional knockout gene OGT mouse model obtained by the method described in any one of claims 1-3.

5. The application of the conditional knockout OGT mouse model as described in claim 4 in screening drugs for osteoarthritis and osteoporosis.

6. The application of the conditional knockout gene OGT mouse model as described in claim 4 in functional studies of skeletal development and bone formation.

7. A kit for constructing an OGT gene conditional knockout animal model, the kit comprising the Cas9 protein, gRNA, donor vector, and primers as shown in SEQ ID NO:4~SEQ ID NO:7 as described in claim 1.

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