Construction method and application of skeletal muscle muscle fiber conditionally induced knockout hnRNPK gene mouse model

Abstract

The invention discloses a construction method and application of a skeletal muscle muscle fiber conditionally induced knockout hnRNPK gene mouse model, and belongs to the technical field of animal models.The construction method comprises the following steps that 1, an hnRNPKflox / flox mouse and an ACTA1-CreEsr1 positive mouse are hybridized, and hnRNPKflox / +-ACTA1-CreEsr1 positive filial generation mice are screened out; (2) hybridizing the hnRNPKflox / +-ACTA1-CreEsr1 positive offspring mice with the hnRNPKflox / flox mice, and screening out the hnRNPKflox / flox-ACTA1-CreEsr1 positive mice, so as to obtain the hnRNPKflox / flox-ACTA1-CreEsr1 positive offspring mice, and screening out the hnRNPKflox / flox-ACTA1-CreEsr1 positive offspring mice, and (3) carrying out Tamoxifen treatment on the hnRNPKflox / flox-ACTA1-CreEsr1 positive mouse, activating Cre recombinase, cutting off the third intron fragment to the seventh intron fragment of the hnRNPK gene, and realizing selective knockout of the hnRNPK gene, namely constructing the mouse model for conditionally inducing and knocking out the hnRNPK gene from the skeletal muscle fiber. According to the invention, the vacancy in preparation of mice with skeletal muscle muscle fiber induced knockout hnRNPK genes in the prior art is filled, and a credible animal model is provided for revealing the functional research of the RNA binding protein hnRNPK in skeletal muscle growth and development regulation at an individual level.

Description

Technical Field

[0001] The present invention relates to the technical field of animal models, and in particular to a method for constructing a skeletal muscle fiber conditionally induced knockout hnRNPK gene mouse model and its application. Background Art

[0002] Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a class of widely expressed RNA-binding proteins (RBPs). This family includes 20 members, ranging from hnRNPA1 to hnRNPU. They have been detected in the nucleus, cytoplasm, plasma membrane and mitochondria, and interact with elements located in the promoter region of genes. hnRNPK is one of the most studied members of the hnRNP family. Its relative molecular weight is about 66 kDa, and it mainly contains four functional domains, of which three K homology domains (KH1, KH2 and KH3) are responsible for binding to single-stranded "UC3-4" RNA and single-stranded or double-stranded "CTTCC" DNA; the KI domain is responsible for binding to specific proteins; in addition, there is an NLS nuclear localization signal sequence and a KNS nuclear cytoplasmic shuttling signal sequence at the N-terminus and C-terminus, respectively.

[0003] The structural characteristics of hnRNPK protein determine the complexity of its function and mechanism of action in cells. Studies have found that hnRNPK, as an important regulatory factor, participates in important biological processes such as muscle development, spermatogenesis, angiogenesis, erythrocyte maturation, organogenesis and cancer by affecting protein expression. - / - Homozygous mice die as early as 13.5 dpc, while hnRNPK + / - Although heterozygotes can be born, their survival rate is extremely low, their tumor incidence rate is high, and their growth and development are significantly delayed, which brings great inconvenience to the study of the function and mechanism of action of hnRNPK in cells.

[0004] The human hnRNPK gene is located at 9q21.32 on chromosome 9. Studies have shown that if the hnRNPK gene base is missing or mutated, it will lead to AKS (atypical Kabuki syndrome), and the patient will have slow growth and development and intellectual disability. In the past decade, researchers have linked abnormal expression of hnRNPK to the progression of cancer, but there are few studies on hnRNPK in myogenic differentiation. In myogenic differentiation, hnRNPK and hnRNPL can interact with MyoD-induced super enhancer RNA (seRNA) to activate the transcription of target genes in the process of myogenic differentiation, thus playing a key role in myogenic differentiation. In addition, in C2C12 myoblasts, hnRNPK interacts with lncRNA Myopar and plays multiple Myopar-related and Myopar-independent roles in the process of myogenic differentiation; and hnRNPK is upregulated during skeletal muscle aging, which may be involved in regulating the demethylation of retrotransposons in aged skeletal muscle.

[0005] The above studies show that the role of hnRNPK in myogenic differentiation is relatively complex and can play a role in multiple ways. It is an important factor in regulating muscle development. - / - Homozygous knockout of mice is lethal, especially systemic knockout of hnRNPK, which results in a very high embryonic lethality rate in mice, making it very difficult to conduct in vivo studies on the function of hnRNPK in skeletal muscle development. Therefore, for those skilled in the art, constructing a conditional knockout mouse model of hnRNPK gene in muscle fibers is crucial to revealing the regulatory mechanism of hnRNPK in skeletal muscle development. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing and applying a mouse model of skeletal muscle fiber conditional inducible knockout of the hnRNPK gene, which fills the gap in the prior art in the preparation of skeletal muscle fiber conditional knockout of the hnRNPK gene mouse, and provides a credible animal model for revealing the function of RNA binding protein hnRNPK in the regulation of skeletal muscle growth and development at the individual level.

[0007] The technical solution adopted by the present invention is as follows:

[0008] One of the purposes of the present invention is to provide a method for constructing a skeletal muscle fiber conditionally induced knockout hnRNPK gene mouse model, comprising the following steps:

[0009] (1) hnRNPK flox / flox Mice were crossed with ACTA1-CreEsr1-positive mice and screened for hnRNPK flox / + -ACTA1-CreEsr1 positive offspring mice;

[0010] (2) hnRNPK flox / + -ACTA1-CreEsr1-positive offspring mice and hnRNPK flox / flox Mice were crossed and hnRNPK was screened flox / flox -ACTA1-CreEsr1 positive mice;

[0011] (3) Using inducers to regulate hnRNPK flox / flox -ACTA1-CreEsr1-positive mice were treated to activate Cre recombinase and remove the 3rd to 7th intron fragments of the hnRNPK gene, thereby achieving selective knockout of the hnRNPK gene, that is, constructing a mouse model of conditional inducible knockout of the hnRNPK gene in skeletal muscle fibers.

[0012] Preferably, the inducing agent is tamoxifen.

[0013] Furthermore, in step (1), hnRNPK is screened out by genotyping method. flox / + -ACTA1-CreEsr1 positive offspring mice, the conditional primers used for identification are:

[0014] F: 5'-CGAGCCGAGAGTAGCAGTTGTA-3';

[0015] R: 5'-AGGTGGACCTGATCATGGAG-3'.

[0016] Furthermore, in step (2), hnRNPK is screened out by genotyping method. flox / flox -ACTA1-CreEsr1 positive mice, the conditional primers used for identification are:

[0017] F: 5'-CGAGCCGAGAGTAGCAGTTGTA-3';

[0018] R: 5'-AGGTGGACCTGATCATGGAG-3';

[0019] hnRNPK_F2: 5'-GTCTCTCGCTCTGTCTTTGTGGC-3';

[0020] hnRNPK_R2: 5'-GGAAGGGCTCAGATTAAGTGGCAA-3'.

[0021] Preferably, the genotype identification methods used in step (1) and step (2) are both PCR methods.

[0022] The second purpose of the present invention is to provide an application of the skeletal muscle fiber conditional induction knockout hnRNPK gene mouse model obtained by the above construction method in studying the regulatory function of skeletal muscle growth and development.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) Based on the characteristics of hnRNPK in myogenic differentiation and considering the - / - Homozygous knockout of mice leads to lethality. The present invention utilizes hnRNPK flox / flox Mice were crossed with ACTA1-CreEsr1-positive mice to obtain hnRNPK flox / + -ACTA1-CreEsr1-positive offspring mice, which were then crossed with hnRNPK flox / flox Mouse hybridization to obtain hnRNPK flox / flox -ACTA1-CreEsr1 positive mice were generated, and finally, the Actal promoter was induced to drive the conditional expression of Cre recombinase in muscle fibers, and the 3rd to 7th intron fragments of the hnRNPK gene were removed, so that hnRNPKmRNA could not be translated normally, thereby selectively knocking out the hnRNPK gene in muscle fibers, and finally obtaining inducible skeletal muscle fiber conditional gene knockout hnRNPK mice. The experimental results showed that the hnRNPK gene was induced to knock out in mature muscle fibers that induced the expression of Cre recombinase, and its genotype was hnRNPK flox / flox -ACTA1-CreEsr1 positive, and the genotype remains hnRNPK in other tissue cells without Cre expression flox / flox , and hnRNPK decreased by more than 40% (p<0.01), and the genotype ratio of the offspring mice conformed to Mendel's law. This shows that the present invention successfully constructed a skeletal muscle fiber conditional induction knockout hnRNPK gene mouse model through reasonable scheme design.

[0025] Therefore, the present invention fills the gap in the prior art in the preparation of skeletal muscle fiber induced knockout hnRNPK gene mice, avoiding the problem that hnRNPK systemic knockout causes individual death and cannot be used for postnatal skeletal muscle development research. And the experimental results also show that after a period of normal feeding, the hnRNPK induced knockout mice are significantly different from normal mice in morphology and size (p<0.01), and the cross-sectional area of ​​the muscle fibers of the mice after hnRNPK induced knockout is reduced. Therefore, the skeletal muscle fiber conditional induced knockout hnRNPK gene mouse model constructed by the present invention is very targeted, and provides a credible animal model for revealing the functional research of RNA binding protein hnRNPK in the regulation of skeletal muscle growth and development at the individual level.

[0026] (2) The present invention designed two pairs of conditional primers and combined them with PCR to respectively target hnRNPK flox / + -ACTA1-CreEsr1-positive offspring mice and hnRNPK flox / flox -ACTA1-CreEsr1 positive mice were identified and screened. The primers designed by the present invention have the advantages of high identification efficiency and accurate screening, and provide a good guarantee for the successful construction of the skeletal muscle fiber conditional induction knockout hnRNPK gene mouse model.

[0027] (3) The scheme of the present invention is reasonably designed and easy to operate. Its various links are closely linked, complementary and related. The constructed skeletal muscle fiber conditional induced knockout hnRNPK gene mouse model has important application prospects in the study of muscle development. Therefore, the present invention is very suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 hnRNPK in the embodiment of the present invention flox / flox Schematic diagram of the mouse construction method.

[0029] Figure 2 The flowchart is a method for constructing a mouse model of conditional induced knockout of hnRNPK gene in skeletal muscle fibers in an embodiment of the present invention.

[0030] Figure 3 hnRNPK in the embodiment of the present invention flox / flox Schematic diagram of PCR identification of homozygous mice, Marker: DL2000 marker.

[0031] Figure 4 Schematic diagram of PCR identification of Cre in mice in the present invention, Marker: DL2000 marker; Acta1-CreEsr1: Cre-positive mice; Acta1 + / + : Cre-negative mice.

[0032] Figure 5 The expression of hnRNPK protein level in TA tissue of WT and hnRNPK-cKO mice was detected by western blot in the examples of the present invention.

[0033] Figure 6 Schematic diagram of grayscale analysis of protein expression in an embodiment of the present invention; n=3, **p<0.01.

[0034] Figure 7 This is a representative schematic diagram of WT and hnRNPK-cKO mice after muscle-induced knockout in the examples of the present invention.

[0035] Figure 8 This is a representative schematic diagram of the skin removed from the muscle of WT and hnRNPK-cKO mice in the examples of the present invention.

[0036] Fig. 9 This is a representative schematic diagram of TA tissues of WT and hnRNPK-cKO mice in the examples of the present invention.

[0037] Fig.10 Schematic diagram showing the comparison of body weights of WT and hnRNPK-cKO mice in the examples of the present invention.

[0038] Fig.11 The muscle tissue weights of WT and hnRNPK-cKO mice in the examples of the present invention; n=6, *p<0.05.

[0039] Fig.12 Schematic diagram of H&E staining results of TA tissues of WT and hnRNPK-cKO mice in the examples of the present invention.

[0040] Fig.13 Schematic diagram of muscle fiber cross-sectional area statistics in the embodiment of the present invention; Bar=100 μm, n=6, **p<0.01. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and embodiments. The embodiments of the present invention include but are not limited to the following embodiments.

[0042] Example

[0043] This example provides a skeletal muscle fiber conditional induction knockout hnRNPK gene mouse model, which is constructed using the Cre-loxp system, as follows:

[0044] 1. Introduction and breeding of mice

[0045] hnPK flox / flox Mouse gene editing commissioned by Saiye Biotechnology Co., Ltd. Technical construction, construction methods such as Figure 1 Acta1-CreEsr1 mice, strain name B6.Cg-Tg(ACTA1-cre / Esr1*)2Kesr / J, were purchased from Jackson Lab in the United States, 2 males and 2 females. The mice express tamoxifen-induced Cre recombinase in the limbs and craniofacial skeletal muscles. The mice were raised in accordance with the SPF animal breeding standards. After isolation and observation without abnormality, they were placed in the breeding area, and the experimental operations were carried out in strict accordance with the relevant regulations of SPF animal management.

[0046] 2. Construction of a mouse model of hnRNPK gene conditionally induced knockout in skeletal muscle fibers

[0047] like Figure 2 As shown, first, hnRNPK flox / flox The mice were crossed with the Acta1-CreEsr1-positive mice to obtain Acta1-CreEsr1-positive-hnRNPK flox / + Mice and Acta1-CreEsr1-negative-hnRNPK flox / + Mice, and then the offspring mice were genotyped and hnRNPK flox / + -ACTA1-CreEsr1 positive offspring mice.

[0048] Next, hnRNPK flox / + -ACTA1-CreEsr1-positive offspring mice and hnRNPK flox / flox Mouse hybridization, including hnRNPK flox / flox -ACTA1-CreEsr1-positive;hnRNPK flox / + -Acta1-CreEsr1 negative; hnRNPK flox / flox and hnRNPK flox / + -ACTA1-CreEsr1 negative. Then genotype identification was performed to screen for hnRNPK flox / flox -ACTA1-CreEsr1-positive mice.

[0049] Finally, for hnRNPK flox / flox -ACTA1-CreEsr1 positive mice were treated with Tamoxifen to activate Cre recombinase and remove the 3rd to 7th intron fragments of the hnRNPK gene, which contains the translation start site, resulting in the inability of hnRNPK mRNA to be translated normally, thus achieving the knockout of the hnRNPK gene. Thus, a mouse model of skeletal muscle fiber conditional induction knockout of the hnRNPK gene was constructed (named hnRNPK-cKO mouse).

[0050] In this example, the PCR method was used to screen and identify the above-mentioned positive mice (wherein hnRNPK flox / flox -ACTA1-CreEsr1 positive mice were identified and screened by double PCR).

[0051] Specifically, the mouse DNA extraction steps are as follows:

[0052] (1) Use sterile scissors to cut off about 2 mm 2 The ears were placed in a sterile 1.5 mL centrifuge tube;

[0053] (2) Add 75 μL of DNA alkaline lysis solution and heat in a constant temperature metal bath at 98°C for 1 h. Shake the ear every 20 min to completely immerse it in the lysis solution. Then cool it to room temperature, add an equal amount of DNA neutralizing solution, and centrifuge at 4000 rpm for 3 min. The supernatant can be used for DNA identification.

[0054] hnPK flox / flox The PCR identification process for homozygous mice is as follows:

[0055] Using mouse whole genome DNA as template, hnRNPK flox / flox The mouse primers were used for PCR amplification and electrophoresis detection. The amplification results showed that the mice with a 473bp band were mice with two Loxp sites, the mice with two bands of 360bp and 473bp were mice with one Loxp site, and the mice with only a 360bp band were wild-type mice (WT) without Loxp sites. The PCR identification results are shown in Figure 3 As you can see, the amplified 473bp band is hnRNPK. flox / flox mouse (lanes 2, 3, 5, 6, 7), and the two bands of 360 bp and 473 bp were hnRNPK flox / + Mouse (lanes 3, 8).

[0056] The specific design of primer sequences is as follows:

[0057] hnRNPK_F2: 5'-GTCTCTCGCTCTGTCTTTGTGGC-3';

[0058] hnRNPK_R2: 5'-GGAAGGGCTCAGATTAAGTGGCAA-3'.

[0059] The identification was performed by conventional PCR amplification method. The PCR reaction conditions were: 98°C, 5 min; 95°C, 30 s, 60°C, 30 s, 72°C, 30 s, 34 cycles; 72°C, 5 min; 12°C, 10 min; and stored at 16°C. The PCR products were subjected to 2% agarose gel electrophoresis. After electrophoresis, the photos were taken with a Bio Rad gel electrophoresis imager.

[0060] The PCR identification process of mouse Cre is as follows:

[0061] Using mouse whole genome DNA as template, PCR amplification electrophoresis detection was performed with Cre mouse primers. PCR amplification electrophoresis detection showed that the amplified 1100bp band corresponded to Acta1-CreEsr1 positive mice, and the blank band corresponded to WT mice. PCR identification results are shown in Figure 4As can be seen, the mice with an amplified 1100bp band are Acta1-CreEsr1 positive mice (lanes 2, 4, 5, and 8), while the blank bands are WT mice (lanes 3, 6, and 7). The results show that the genotype ratio of the offspring mice conforms to Mendel's law.

[0062] The specific design of primer sequences is as follows:

[0063] F (upstream primer): 5′-CGAGCCGAGAGTAGCAGTTGTA-3′;

[0064] R (downstream primer): 5'-AGGTGGACCTGATCATGGAG-3'.

[0065] The identification was performed by conventional PCR amplification method, and the PCR reaction conditions were: 94°C, 3 min; 94°C, 30 s, 62°C, 35 s, 72°C, 35 s, 35 cycles; 72°C, 5 min, 12°C, 10 min; stored at 10°C. The PCR products were subjected to 1% agarose gel electrophoresis.

[0066] Based on the above primers, hnRNPK was identified and screened by PRC. flox / + -ACTA1-CreEsr1 positive offspring mice, the conditional primers used for identification are:

[0067] F: 5'-CGAGCCGAGAGTAGCAGTTGTA-3';

[0068] R: 5'-AGGTGGACCTGATCATGGAG-3'.

[0069] Duplex PCR was used to identify and screen hnRNPK flox / flox -ACTA1-CreEsr1 positive mice, the conditional primers used for identification are:

[0070] F: 5'-CGAGCCGAGAGTAGCAGTTGTA-3';

[0071] R: 5'-AGGTGGACCTGATCATGGAG-3';

[0072] hnRNPK_F2: 5'-GTCTCTCGCTCTGTCTTTGTGGC-3';

[0073] hnRNPK_R2: 5'-GGAAGGGCTCAGATTAAGTGGCAA-3'.

[0074] The knockout efficiency of hnRNPK-cKO mice was tested below. The specific testing process is as follows:

[0075] (1) Tamoxifen was taken at 20 mg mL -1 The ratio of 1:1 was dissolved in corn oil, shaken overnight at 37°C, and stored at 4°C in the dark after complete dissolution. 8-week hnRNPK-cKO mice were selected as experimental mice, and other male mice of the same sex in the same litter were selected as control mice. Tamoxifen was injected intraperitoneally for 5 consecutive days (1 time / d), 100 μL each time.

[0076] (2) Two weeks after the induction, the experimental mice were killed by cervical dislocation, and the TA muscles of the mice were obtained using dissection tools. The collected TA tissues were subjected to protein extraction and Western blot to detect the expression of hnRNPK protein levels in the muscle tissue. The protein extraction process was as follows: the tissues were ground and lysed on an automatic sample grinder, placed on ice for 15 minutes, placed on ice for lysis, and the cells were repeatedly lysed for about 1 hour, followed by centrifugation at 4°C and 12,000 g for 20 minutes. The supernatant was aspirated as much as possible, and the protein concentration was finally detected using the BCA protein concentration assay kit (Biyuntian). After adding the protein loading buffer in proportion, the protein was boiled at 100°C for 5 minutes to denature the protein.

[0077] The Western blot process is as follows: wash the glass plate, add 12% concentrated gel to the glass plate, add 5% separation gel after the concentrated gel solidifies, add 15ug of denatured protein to each well after the SDS-PAGE gel solidifies; first use 60V, 30min constant voltage electrophoresis on the concentrated gel, and switch to 120V when the sample is electrophoresed to the separation gel until the sample is electrophoresed to the bottom of the separation gel, and the electrophoresis ends; cut the PVDF membrane (Millipore) according to the gel size, activate it with methanol for 5s before use, and transfer it to the membrane at a constant current of 200mA for 1.5-2h; use 5% skim milk powder and block it at room temperature for 2h; incubate with primary antibody at 4℃ overnight; wash the membrane twice with TBST and once with TBS, each time for 10min. Add secondary antibody and incubate at room temperature for 2h, wash the membrane twice with TBST and once with TBS; color reaction of PVDF membrane is carried out, and the chemiluminescence imager is exposed to take pictures.

[0078] The results are as follows Figure 5 , Figure 6 As shown, hnRNPK in the experimental group was reduced by more than 40% (p<0.01), which indicated that the knockout strategy induced by TMX was effective and that the knockout mouse model was successfully constructed.

[0079] The following is a verification of the effect of hnRNPK on mouse skeletal muscle development.

[0080] Based on the continuous feeding of hnRNPK-cKO mice, it was found that severe spinal curvature appeared 15 days after the induction of knockout, while the control mice were normal, indicating that hnRNPK is essential for the development of mouse muscle fibers (e.g. Figure 7 shown).

[0081] The details are as follows:

[0082] First, 14 days after hnRNPK was induced and knocked out, the mice were sampled and analyzed. The experimental mice were killed by cervical dislocation, and the TA, EDL, SOL, and GAS tissues of the mice were taken using dissection tools. The tissues were weighed with an electronic balance, the data were recorded, and photos were taken for observation. Figure 8-Figure 11 As shown, the body weight of mice was significantly reduced after hnRNPK induced knockout (p<0.05), the color of the skinned mice muscles was significantly darker than that of the control group, the weight of the tibialis anterior (TA), soleus (SOL) and gastrocnemius (GAS) muscles were also significantly lower than those of the control group (p<0.05), and there was no difference in the weight of the extensor digitorum longus (EDL) (p>0.05).

[0083] Then, the mouse tibialis anterior muscle (TA) was collected and stored in 4% paraformaldehyde for H&E staining of tissue sections. The H&E staining process is as follows:

[0084] Dewaxing: Take paraffin sections and dewax them in xylene I for 10 min, then dewax them in xylene II for 10 min;

[0085] Rehydration: Rehydrate in 100% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 3 min each, and wash with deionized water for 2 min;

[0086] Hematoxylin staining: Take the cleaned sections and stain them with hematoxylin staining solution for 5 minutes. After staining, rinse off the excess staining solution with tap water and wash once with distilled water;

[0087] Eosin staining: Take the above sections and stain with eosin staining solution for 2 minutes. Rinse off the excess staining solution with tap water;

[0088] Dehydration: Place the stained sections in 70%, 80%, 90%, and 100% ethanol for dehydration, with the dehydration time being 2 minutes each.

[0089] Transparentization: Place the dehydrated slices in xylene for 5 minutes, then take them out and place them in new xylene and repeat the operation once;

[0090] Sealing: Take out the slices, dry them, seal them with neutral resin, and observe and photograph them under an inverted microscope.

[0091] The results are as follows Fig.12 , Fig.13As shown, the induced knockout group was significantly different from the control group mice in morphology and size (p<0.01), and the muscle fiber cross-sectional area was reduced by about 30%, indicating that knocking out hnRNPK inhibited muscle development by inhibiting muscle fiber cross-sectional area.

[0092] in conclusion:

[0093] 1. The present invention successfully constructed a mouse model of skeletal muscle fiber conditional inducible knockout of hnRNPK gene, filling the gap in the prior art in the preparation of skeletal muscle fiber conditional inducible knockout of hnRNPK gene mouse.

[0094] 2. According to the feeding conditions of the constructed mouse model, hnRNPK is essential for the development of mouse muscle fibers, and knocking out hnRNPK inhibits muscle development by inhibiting the cross-sectional area of ​​muscle fibers. Therefore, the present invention provides a very important reference for revealing the functional research of RNA binding protein hnRNPK in the regulation of skeletal muscle growth and development at the individual level and the formulation of related strategies.

[0095] The above embodiments are only preferred implementation modes of the present invention and should not be used to limit the protection scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention and have no substantive significance, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention.

Claims

1. A method for constructing a mouse model of skeletal muscle fiber conditional induction knockout of hnRNPK gene, characterized in that: The following steps are involved: (1) hnRNPK flox / flox Mice were crossed with ACTA1-CreEsr1-positive mice and screened for hnRNPK flox / + -ACTA1-CreEsr1 positive offspring mice; (2) hnRNPK flox / + -ACTA1-CreEsr1-positive offspring mice and hnRNPK flox / flox Mice were crossed and hnRNPK was screened flox / flox -ACTA1-CreEsr1 positive mice; (3) Using inducers to regulate hnRNPK flox / flox -ACTA1-CreEsr1-positive mice were treated to activate Cre recombinase and remove the 3rd to 7th intron fragments of the hnRNPK gene, thereby achieving selective knockout of the hnRNPK gene, that is, constructing a mouse model of conditional inducible knockout of the hnRNPK gene in skeletal muscle fibers.

2. The method for constructing a skeletal muscle fiber conditionally induced knockout mouse model of hnRNPK gene according to claim 1, characterized in that: The induction agent is tamoxifen.

3. The method for constructing a skeletal muscle fiber conditionally induced hnRNPK gene knockout mouse model according to claim 1 or 2, characterized in that: In the step (1), hnRNPK is screened by genotyping method. flox / + -ACTA1-CreEsr1 positive offspring mice, the conditional primers used for identification are: F: 5'-CGAGCCGAGAGTAGCAGTTGTA-3'; R: 5'-AGGTGGACCTGATCATGGAG-3'.

4. The method for constructing a skeletal muscle fiber conditionally induced knockout mouse model of hnRNPK gene according to claim 3, characterized in that: In the step (2), hnRNPK is screened by genotyping method. flox / flox -ACTA1-CreEsr1 positive mice, the conditional primers used for identification are: F: 5'-CGAGCCGAGAGTAGCAGTTGTA-3'; R: 5'-AGGTGGACCTGATCATGGAG-3'; hnRNPK_F2: 5'-GTCTCTCGCTCTGTCTTTGTGGC-3'; hnRNPK_R2: 5'-GGAAGGGCTCAGATTAAGTGGCAA-3'.

5. The method for constructing a skeletal muscle fiber conditionally induced hnRNPK gene knockout mouse model according to claim 4, characterized in that: The genotype identification methods used in step (1) and step (2) are both PCR methods.

6. Use of the skeletal muscle fiber conditional inducible knockout hnRNPK gene mouse model obtained by the construction method according to any one of claims 1 to 5 in studying the regulatory function of skeletal muscle growth and development.

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