POMGnT1 gene knockout mouse model as well as construction method and application thereof

Abstract

The invention discloses a POMGnT1 gene knockout mouse model and a construction method and application, and relates to the technical field of gene knockout, the method comprises the following steps: S1, obtaining a floxp mouse of which a POMGnT1 gene is conditionally knocked out; s2, a CaMK2 alpha-Cre tool mouse is obtained; s3, hybridizing the heterozygote floxp mouse with a CaMK2 alpha-Cre tool mouse, so as to obtain a CaMK2 alpha-Cre / POMGnT1f / + gene mouse; and hybridizing a CaMK2 alpha-Cre / POMGnT1f / + gene mouse and a homozygote floxp mouse or mating a male CaMK2 alpha-Cre / POMGnT1f / + gene mouse and a female CaMK2 alpha-Cre / POMGnT1f / + gene mouse, and screening to obtain the mouse model of which the POMGnT1 gene is specifically knocked out. According to the scheme, the POMGnT1 gene knockout mouse can be constructed, so that the influence of POMGnT1 on the structure and function of the brain can be explored.

Description

Technical Field

[0001] The present invention relates to the technical field of gene knockout, and in particular to a POMGnT1 gene knockout mouse model, a construction method and an application thereof. Background Art

[0002] Post-translational modifications (PTMs) of proteins are chemical modifications that promote the increase of complexity from the genome level to the proteome. The post-translational modifications of proteins in higher eukaryotes mainly include methylation, ubiquitination, acetylation, glycosylation, phosphorylation, etc. Among them, glycosylation is one of the most common PTMs. It can increase the heterogeneity and functional diversity of proteins, participate in signal transduction, transcription and cytoskeleton stability, and is related to the occurrence of neurodegenerative diseases. In recent years, it has been found that protein glycosylation plays an important role in the development of the central nervous system. For example, O-GlcNAc glycosylation modification can regulate the function of neurons; N-glycosylation modification can regulate the differentiation of neural stem cells and brain development; insufficient mannosylation modification can lead to congenital muscular dystrophy, severe muscle atrophy and abnormal brain development.

[0003] Mannosylation is an essential glycosylation modification in mammals, and O-mannose accounts for 30% of the known O-linked polysaccharides in the brain. In vivo, the formation of mannosylation requires the participation of mannosyltransferases, and protein-O-linked mannose B1,2-N-acetylglucosaminyl-transferasel 1 (POMGnT1) is a mannosyltransferase that has been studied in depth. The expression of POMGnT1 mRNA in different tissues such as human gray matter, white matter, cerebellum, spinal cord, diaphragm, heart, pancreas, liver and kidney was analyzed by reverse transcriptase chain reaction. The results showed that the transcripts of POMGnT1 were expressed in various tissues including the central nervous system and skeletal muscle. In normal mouse brain tissue, the transcripts and protein products of POMGnT1 are strongly expressed in the cerebral cortex and hippocampus; POMGnT1 protein is mainly expressed in mature neurons; at the organelle level, POMGnT1 is mainly enriched in the Golgi apparatus. It can be seen that the expression of POMGnT1 protein in the mouse brain has certain regional specificity and cell specificity.

[0004] POMGnT1 has the characteristics of a classic mannosyltransferase. It can catalyze the transfer of N-acetyl-glucosamine groups (GICNAc) to mannose linked to the oxygen position of glycoproteins. The deficiency of this enzyme will lead to insufficient mannosylation, and cognitive dysfunction in adulthood is a prominent manifestation. The mutation of POMGnT1 will affect the post-translational modification of certain proteins in brain tissue, such as dystroglycan (DG). POMGnT1 can increase the mannose moiety of the α-subunit of DG located outside the cell, thereby promoting the connection between DG and the basement membrane (BM) of the extracellular matrix. The binding force of α-DG with insufficient mannosylation modification to the laminin and collagen of the extracellular matrix is ​​weakened. POMGnT1 gene mutations lead to muscle-eye-brain (MEB) disease, one of the main characteristics of which is anatomical abnormalities in the brain. These findings suggest that POMGnT1 may affect brain development in vivo by mannosylation of α-DG. Among the tissues / organs of higher animals, the brain is the most functionally complex part, and the regulation of its protein expression is also the most complex and delicate. This specific expression feature of POMGnT1 protein and its effect on mannosylation modification suggest that POMGnT1 protein plays an important role in brain development.

[0005] Considering the specific expression characteristics of POMGnT1 in the brain and its effects on brain structure and function, this study constructed brain-specific POMGnT1 gene knockout mice (prefrontal lobe and hippocampus knockout) to further explore the effects of POMGnT1 on brain structure and function during brain development in mice. Summary of the invention

[0006] To address the deficiencies of the prior art, the present invention provides a POMGnT1 gene knockout mouse model and a construction method and application thereof, which can construct a POMGnT1 gene knockout mouse model for in-depth research on brain structure and function.

[0007] In order to achieve the purpose of the present invention, the following scheme is proposed: A method for constructing a POMGnT1 gene knockout mouse model comprises the following steps: S1. Using CRISPR / Cas9 technology, Cas9 mRNA, gRNA and targeting vector were electroporated into mouse fertilized eggs, which were then transplanted into surrogate mice to produce F0 generation, and floxp mice with conditional knockout of POMGnT1 gene were screened; S2, obtain CaMK2α-Cre tool mice; S3. Hybridize heterozygous floxp mice with CaMK2α-Cre mice to obtain CaMK2α-Cre / POMGnT1 f / + Genetic mouse; CaMK2α-Cre / POMGnT1 f / + Genetic mice were crossed with homozygous floxp mice or male and female CaMK2α-Cre / POMGnT1 f / + The transgenic mice were mated and a mouse model with specific POMGnT1 gene knockout was obtained.

[0008] Furthermore, in step S1, the screening and identification primers for floxp mice with conditional knockout of POMGnT1 gene include: POMGnT1-floxp-F:5'-ACTTCCTAATCCCTTTGGCTTCA-3'; POMGnT1-floxp-R:5'-CCATCCACCTTTTAACACTTCCCT-3'.

[0009] A POMGnT1 gene knockout mouse model is obtained by the construction method.

[0010] The POMGnT1 gene knockout mouse model is used in the field of psychiatric and neurological diseases. The POMGnT1 gene knockout mouse model is used for detecting degenerated neurons.

[0011] The POMGnT1 gene knockout mouse model is used in the field of psychiatric and neurological diseases. The POMGnT1 gene knockout mouse model is used for detecting cell necrosis in the prefrontal lobe and hippocampus.

[0012] The beneficial effects of the present invention are: a specific POMGnT1 gene knockout mouse can be constructed to further explore the influence of POMGnT1 on the structure and function of the brain; the POMGnT1 gene knockout mouse can be used for the detection of degenerated neurons, and the results show that the number of degenerated neurons in the hippocampus and prefrontal lobe of the mice increases, indicating that the brain neurons of the mice are aging prematurely; the POMGnT1 gene knockout mouse can be used for the detection of cell necrosis in the prefrontal lobe and hippocampus, and the results show that obvious pathological changes occur in the CA3 region of the mice, and necrosis occurs in the pyramidal cells of the region and the hippocampus. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the process for constructing the POMGnT1 cKO mouse model; Figure 2 Electropherogram for PCR identification of POMGnT1 cKO mice; Figure 3A is the FJC staining image of the prefrontal cortex and hippocampus of 10M POMGnT1 cKO mice, and B is the FJC staining image of the prefrontal cortex and hippocampus of 15M POMGnT1 cKO mice; Figure 4 A in the middle is the HE staining image of the mouse hippocampus and prefrontal cortex, and B is the Nissl staining image of the mouse hippocampus and prefrontal cortex. DETAILED DESCRIPTION

[0014] Example 1 This embodiment provides a method for constructing a POMGnT1 gene knockout mouse model, comprising the following steps: S1 and POMGnT1 are located on chromosome 4 of mice. Using CRISPR / Cas9 technology, Cas9 mRNA, gRNA and targeting vector were electroporated into mouse fertilized eggs, which were then transplanted into surrogate mice to produce F0 generations, and floxp mice with conditional knockout of POMGnT1 gene were screened. Among them, the screening and identification primers include: POMGnT1-floxp-F:5'-ACTTCCTAATCCCTTTGGCTTCA-3'; POMGnT1-floxp-R:5'-CCATCCACCTTTTAACACTTCCCT-3'.

[0015] S2. Obtain CaMK2α-Cre tool mice. The CaMK2α-Cre tool mice express Cre enzyme only in neurons that specifically express CaMK2α protein. Therefore, Cre enzyme is only specifically expressed in specific areas of the brain tissue, such as the prefrontal cortex and hippocampus. Therefore, the mouse model for knocking out the POMGnT1 gene specifically knocks out the POMGnT1 gene only in the prefrontal cortex and hippocampus.

[0016] It should be noted here that the above-mentioned floxp mice and CaMK2α-Cre tool mice were provided by Saiye Biotechnology Co., Ltd.

[0017] S3, such as Figure 1 As shown, heterozygous floxp mice were crossed with CaMK2α-Cre mice to obtain CaMK2α-Cre / POMGnT1 f / + Genetic mouse; CaMK2α-Cre / POMGnT1 f / + Genetic mice were crossed with homozygous floxp mice or male and female CaMK2α-Cre / POMGnT1 f / + Genetic mice were mated to screen for a mouse model with specific knockout of the POMGnT1 gene, namely POMGnT1cKO mice, or CaMK2α-Cre / POMGnT1 f / f Genetic mouse.

[0018] Example 2 This example provides a method for identifying a mouse model with specific knockout of the POMGnT1 gene, and the specific steps are as follows: Use TaKaRa Mini BEST Universal Genomic DNA Extraction Kit (Ver.5.0 Code No. 9765) to obtain high-purity genomic DNA: a. Take 2mm-5mm mouse tissue and place it in a 1.5ml centrifuge tube; b. Add 180 μL Buffer GL, 20 μL Proteinase K and 10 μL RNase-A to the centrifuge tube and incubate in a 56°C water bath overnight until the tissue is completely dissolved; c. Add 200 μl Buffer GB and 200 μl 100% ethanol to the lysate and mix thoroughly by pipetting; d. Place the adsorption column on the collection tube, transfer the solution to the adsorption column, centrifuge at 12000rpm for 2min, and discard the filtrate; e. Add 500 μl of Buffer WA to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the filtrate; f. Add 700μl of Buffer WB to the adsorption column, centrifuge at 12000rpm for 1min, and discard the filtrate; make sure that the specified volume of 100% ethanol has been added to Buffer WB; add Buffer WB around the wall of the adsorption column, which will help to completely wash away the salt adhering to the wall; g. Repeat step d; h. Place the adsorption column on the collection tube and centrifuge at 12000 rpm for 2 min; i. Place the adsorption column on a new 1.5ml centrifuge tube, add 50μl-200μl of sterile water or Elution Buffer to the center of the adsorption column membrane, and let it stand at room temperature for 5 minutes; note that the sterile water or Elution Buffer should be heated to 65℃ before use, which is conducive to improving the elution efficiency; j. Centrifuge at 12000rpm for 2min to elute DNA. If a larger yield is required, add the centrifuge solution back to the center of the adsorption column membrane or add 50μl-200μl of sterile water or Elution Buffer. After standing at room temperature for 5min, centrifuge at 12000rpm for 2min to elute DNA. k. Measure the DNA concentration using a DNA concentration meter; l. Prepare the PCR mixture according to the following system: m. PCR reaction system: n. Prepare TAE solution: first dissolve TAE powder in 600ml ddH2O, stir with a magnetic stirrer, and then dilute to 1L TAE solution with ddH2O; o. Prepare agarose solution: 1g agar is dissolved in 100ml TAE fresh solution, which can be used to prepare the whole gel; (1 / 2 gel: 50ml agar solution, 1 / 4 gel: 25ml agar solution) 1) Weigh 1g agar powder Agrose and place it in a beaker; 2) Pour in 100ml TAE solution; 3) Heat in a microwave oven to dissolve the agar until it is transparent, take it out after heating for 20s each time, shake the beaker, and repeat until the solution is transparent; 4) After the agar solution is cooled to a temperature that is not hot when placed on the back of the hand, add 10μl or 20μl nucleic acid fluorescent dye GoodView, and be careful to avoid light; 5) Shake to mix; p. Gel preparation: After cooling and solidification, pour in TAE solution and take out and put into the electrophoresis tank, with the sample well facing the black electrode; q. Sample loading: DNA Marker: 3μl, Sample: 10μl; r. Electrophoresis: 110V, 37min; s. Development.

[0019] The results are as follows Figure 2 As shown, POMGnT1 in POMGnT1 cKO mice was cleaved at the genome level, and POMGnT1 was knocked out at the genome level.

[0020] Example 3 Detection of degenerating neurons in POMGnT1 cKO mice The Solarbio-G3262 degenerative neuron detection kit was used to detect the activity of degenerated neurons in POMGnT1 cKO mice. The experimental steps were performed according to the instructions of the kit. Only when neurons are degenerated will they be stained green, and normal neurons are not stained. Therefore, it is possible to determine whether the brain cells are degenerated based on the staining results of the kit, and then take photos under the same parameters with a microscope and count the number of positive cells. The results are shown in Figure 2. Figure 3 Figure A shows the FJC staining of the prefrontal lobe and hippocampus of 10MPOMGnT1 cKO mice, and Figure B shows the FJC staining of the prefrontal lobe and hippocampus of 15M POMGnT1 cKO mice. It can be seen from the figures that the number of degenerating neurons in the hippocampus and prefrontal lobe of POMGnT1 cKO mice increased, indicating that the brain neurons of POMGnT1 cKO mice aged prematurely.

[0021] Example 4 Pathological Detection of POMGnT1 cKO Mice Solebol G1120-HE and Solebol G1436-Nissl staining were used to detect brain pathological changes in POMGnT1 cKO mice. The experimental steps were performed according to the instructions of the kit. The results are shown in Figure 4 Pathological analysis of the prefrontal cortex and hippocampus of 10M POMGnT1 cKO mice revealed obvious pathological changes in the CA3 region of the mice, with necrosis of pyramidal cells in this region and the hippocampus.

[0022] The above embodiments are only used to illustrate the technical ideas and features of the present invention, and are not intended to be the only or limit the present invention. It should be understood by those skilled in the art that various changes or equivalent substitutions made to the present invention without departing from the scope of the present invention all fall within the scope of protection of the present invention.

Claims

1. A method for constructing a POMGnT1 gene knockout mouse model, characterized in that: The following steps are involved: S1. Using CRISPR / Cas9 technology, Cas9 mRNA, gRNA and targeting vector were electroporated into mouse fertilized eggs, which were then transplanted into surrogate mice to produce F0 generation, and floxp mice with conditional knockout of POMGnT1 gene were screened; S2, obtain CaMK2α-Cre tool mice; S3. Hybridize heterozygous floxp mice with CaMK2α-Cre mice to obtain CaMK2α-Cre / POMGnT1 f / + Genetic mouse; CaMK2α-Cre / POMGnT1 f / + Genetic mice were crossed with homozygous floxp mice or male and female CaMK2α-Cre / POMGnT1 f / + The transgenic mice were mated and a mouse model with specific knockout of the POMGnT1 gene was obtained.

2. The method for constructing a POMGnT1 gene knockout mouse model according to claim 1, characterized in that: In step S1, the screening and identification primers for floxp mice with conditional knockout of POMGnT1 gene include: POMGnT1-floxp-F:5'-ACTTCCTAATCCCTTTGGCTTCA-3'; POMGnT1-floxp-R:5'-CCATCCACCTTTTAACACTTCCCT-3'.

3. A POMGnT1 gene knockout mouse model, characterized in that: Obtained by the construction method described in claim 1 or 2.

4. Use of the POMGnT1 gene knockout mouse model according to claim 3 in the field of psychiatric and neurological diseases, characterized in that: The POMGnT1 gene knockout mouse model is used for detecting degenerating neurons.

5. Use of the POMGnT1 gene knockout mouse model according to claim 3 in the field of psychiatric and neurological diseases, characterized in that: The POMGnT1 knockout mouse model was used to detect cell necrosis in the prefrontal cortex and hippocampus.

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