A method for constructing a testicular MYH11 gene knockout mouse model and its application

Abstract

The present invention relates to the field of genetic engineering technology, and in particular to a method for constructing a testicular MYH11 gene knockout mouse model and its application. The construction method comprises: constructing a targeted homologous recombination vector, introducing the targeted homologous recombination vector, gRNA and Cas9 protein into mice; the 5'-loxP site insertion region in the targeted homologous recombination vector is located in intron 2, and the 3'-loxP site insertion region is located in intron 3. The knockout method provided by the present invention for a specific target can effectively achieve the knockout of the MYH11 gene in the mouse testis, with an off-target rate of 0, which is of great significance in the study of MYH11 gene-related diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a method for constructing a testicular MYH11 gene knockout mouse model and an application thereof. Background Art

[0002] Smooth muscle myosin heavy chain (SMMHC) is encoded by the Myh11 gene, located on chromosome 16, and consists of 1972 amino acids. SMMHC is also known as "SM-MHC" (smooth muscle-myosin heavy chain, SM-MHC) or "MYH11" (myosin heavy polypeptide 11, MYH11). MYH11 is an important marker in the contraction process of vascular smooth muscle cells (VSMCs), and plays an important role in muscle physiology and smooth muscle contraction function. Its selective overexpression can accelerate the contraction rate of airway smooth muscle, thereby causing respiratory diseases such as asthma. Myh11 gene mutations can cause many tumor-related diseases. For example, missense mutations and frameshift mutations of the Myh11 gene can lead to the formation of intestinal tumors and abdominal aortic aneurysms.

[0003] Spermatogenesis is a complex differentiation process that depends largely on the dynamic changes of the cytoskeleton and the role of motor proteins. The cytoskeleton system controls the morphology of cells and is composed of microfilaments, microtubules and intermediate filaments. Myosin is an ATP hydrolase that can convert the chemical energy on ATP into mechanical energy to generate the energy required for movement. At the same time, Myosin is involved in acrosome formation, vesicle transport, gene transcription and nuclear formation. Actin and Myosin cooperate to promote spermatogenesis during spermatogenesis. They influence each other and jointly control the process of cell migration. The walls of male seminiferous tubules contain contractile smooth muscle-like peritubular cells, which are considered to be important cells for sperm transport. Studies have shown that the loss of MYH11 in the peritubular cells of most males with impaired spermatogenesis will lead to a decrease in the contractile ability of the peritubular cells of the testis, thereby reducing male fertility. Therefore, MYH11 plays an important role in the development of the male reproductive system. Myh11 is formed by the selective splicing of exons of dynein, and provides energy for cell movement through ATP hydrolysis and changes in the domain. The destruction of any domain will affect the movement and contraction function of the cell.

[0004] Myosin is a multifunctional protein that participates in cell migration by binding to microfilaments, microtubules and calcium-binding proteins, and provides energy for cell activities through ATPase hydrolysis and structural changes. If the MYH11 gene is knocked out systemically through gene editing, the probability of obtaining homozygous or even heterozygous mice will be very low. Conditional knockout technology can improve the survival rate of gene-edited mouse offspring. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a method for constructing a testicular MYH11 gene knockout mouse model and its application.

[0006] In a first aspect, the present invention provides a method for constructing a testicular MYH11 gene knockout mouse model, comprising:

[0007] constructing a targeted homologous recombination vector, and introducing the targeted homologous recombination vector, gRNA and Cas9 protein into mice;

[0008] In the targeted homologous recombination vector, the 5'-loxP site insertion region is located in intron 2, and the 3'-loxP site insertion region is located in intron 3.

[0009] Furthermore, the insertion position of the 5'-loxP site can be any site in intron 2 of the MYH11 gene, and the insertion position of the 3'-loxP site can be any site in intron 3 of the MYH11 gene.

[0010] Furthermore, the targeting region of the targeted homologous recombination vector includes the nucleotide sequence shown in SEQ ID NO. 1. The targeting region is the region between the 5'-loxP site and the 3'-loxP site.

[0011] Furthermore, the gRNA comprises:

[0012] gRNA1:5'-CAGGGGTCCTAACCTTATATAGG-3',

[0013] gRNA2:5'-TGTGTGCGCTGAAGAGGTACTGG-3'.

[0014] Furthermore, the introduction into mice is:

[0015] The floxed mouse is introduced into a fertilized mouse egg to develop into a floxed mouse, and the floxed mouse is mated with a mouse whose testicular tissue specifically expresses the Vasa-Cre enzyme to obtain an F1 generation of floxed heterozygous mice.

[0016] In a second aspect, the present invention provides a primer combination, comprising:

[0017] Upstream homology arm amplification primer pair:

[0018] F1:5'-AGGAGCATCGGGTCCTCATTTAT-3',

[0019] R1: 5'-GTGGATTCGGACCAGTCTGA-3'; and / or,

[0020] Downstream homology arm amplification primer pair:

[0021] F2:5'-ACGTAAACGGCCACAAGTTC-3',

[0022] R2: 5'-CGTGAAATGGGCATAGTCAAACT-3'; and / or,

[0023] Primer pair for loxP site detection:

[0024] F3:5'-TTTCAGCGGCATCTCTAGTTGT-3',

[0025] R3:5'-TTGCTCATTCACAGTCACATAGCC-3'.

[0026] The present invention further provides a kit comprising the primer combination.

[0027] The present invention further provides the primer combination or the use of the kit in constructing a MYH11 gene knockout mouse model;

[0028] The present invention further provides the construction method or the application of the primer combination in constructing a male reproductive defect mouse model and a urinary system disease mouse model.

[0029] The present invention has the following beneficial effects:

[0030] The present invention discloses a method and application for constructing a Myh11 gene testicular tissue conditional knockout mouse model, wherein the method uses CRISPR / Cas9 gene editing technology to target and knock out exon 3 of the Myh11 gene, and simultaneously utilizes the Cre-loxP recombination system, adopts approaches such as fertilized egg preference homologous recombination repair, improves homologous recombination efficiency, and realizes conditional gene knockout of tissues and cells. For the constructed Myh11 gene knockout mouse, sequencing found that exon 3 deletion mutation and non-homologous recombination repair occurred in the target position of its sequence in the testis. The heterozygous model mouse has the phenotype and characteristics of male testicular development defects and bladder tissue development defects, and can be used to study the mechanism of male reproductive defect diseases and urinary system diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a ratio distribution diagram of the four bases in the CKO region provided in Example 1 of the present invention.

[0032] Figure 2 This is a schematic diagram of the similarity analysis results of BLAST comparison of the upstream 3000bp and downstream 3000bp sequences of the CKO region provided in Example 1 of the present invention in the mouse genome.

[0033] Figure 3 Schematic diagram of the construction of the knockout target site provided in Example 1 of the present invention.

[0034] Figure 4 This is a construction map of the internal homology arms and CKO of the homologous recombination vector provided in Example 1 of the present invention.

[0035] Figure 5 This is the base sequence map of the internal 5' homology arm of the homologous recombination vector provided in Example 1 of the present invention.

[0036] Figure 6 This is the 3' homology arm base sequence map provided in Example 1 of the present invention.

[0037] Figure 7 This is a flowchart for constructing the knockout mice provided in Example 1 of the present invention.

[0038] Figure 8 Schematic diagram of the primer design strategy provided in Example 1 of the present invention.

[0039] Fig. 9 The electrophoresis diagram of the PCR products of the F1 and R1 primers provided in Example 1 of the present invention; wherein, lanes 1, 2, 8 and 21 are the results of PCR using the genome of the F2 generation flox mice as a template, lane M is a molecular marker, lane WT is a control group of wild-type mice, and lane Water is a negative control group of PCR using sterile water as a template.

[0040] Fig.10 The electrophoresis diagram of the PCR products of the F2 and R2 primers provided in Example 1 of the present invention; wherein, lanes 1, 2, 8 and 21 are PCR products using the genome of the F2 generation flox mice as a template, lane M is a molecular marker, lane WT is a control group of wild-type mice, and lane Water is a PCR negative control group using sterile water as a template.

[0041] Fig.11 This is a schematic diagram of the PCR sequencing results provided in Example 1 of the present invention.

[0042] Fig.12 Schematic diagram of the primer design strategy for knockout mouse genotype identification provided in Example 1 of the present invention.

[0043] Fig.13 This is the F5+R5 primer PCR electrophoresis diagram provided in Example 1 of the present invention; wherein lane 15 is the PCR result of heterozygous mice (Flox / +, Vasa-Cre).

[0044] Fig.14 This is the electrophoresis diagram of the Vasa-MF+Vasa-MR primer PCR provided in Example 1 of the present invention; wherein lane 15 is the PCR result of the heterozygous mouse (Flox / +, Vasa-Cre).

[0045] Fig.15 Schematic diagram of the design strategy of Southern Blot detection provided in Example 1 of the present invention.

[0046] Fig.16 This is a diagram showing the results of the Southern Blot probe analysis provided in Example 1 of the present invention.

[0047] Fig.17 This is a graph showing the results of sperm motility analysis of CKO heterozygous male mice provided in Example 1 of the present invention.

[0048] Fig.18 This is a HE staining image of a testicular tissue section provided in Example 1 of the present invention.

[0049] Fig.19 This is a HE staining image of the bladder tissue section provided in Example 1 of the present invention.

[0050] Fig. 20 HE staining images of lung tissue, thoracic aorta and muscle tissue sections provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0051] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0052] Example 1

[0053] 1. Design of MYH11 gene gRNA target sequence and vector construction

[0054] The MYH11 gene (NCBI reference sequence: NM_001161775; ENSEMBL: ENSMUSG00000018830) is located on mouse chromosome 16 and has 41 exons (transcript: ENSMUST00000090287). The present invention found that exon 3 is located in the 5.85% region of the coding region, and its deletion will cause a frameshift mutation in the MYH11 gene; and the exon 3 region of the MYH11 gene does not belong to other genes, and will not cause sequence deletions in other genes after knockout; therefore, exon 3 of the MYH11 gene is evaluated to be the site of action with the highest knockout efficiency and the lowest off-target probability, and is selected as the conditional knockout region (CKO region), and the evaluation data is shown in Figure 1 and Figure 2 . Figure 2 The ratio distribution of the four bases in the CKO region. The sequences of the homology arms and CKO region were analyzed to determine the GC content. No significant high GC content region was found. Therefore, this region is suitable for PCR screening and sequencing analysis. The total length of CKO is 7157bp. Base A (24.27% 1737) | Base C (22.15% 1585) | Base T (27.74% 1985) | Base G (25.85% 1850). Figure 3 Similarity analysis table of BLAST alignment of 3000 bp sequences upstream and downstream of CKO region with mouse genome: 3000 bp upstream and 3000 bp downstream of exon 3 were not found to have homology with other regions of the genome. Figure 3 , the construction map of homology arms and CKO can be found in Figure 4 Deletion of the CKO region should result in loss of function of the mouse MYH11 gene.

[0055] (1) Design gRNA at both ends of exon 3, the sequence is as follows (SEQ ID NO.2-3):

[0056] gRNA1: 5'-CAGGGGTCCTAACCTTATATAGG-3',

[0057] gRNA2: 5'-TGTGTGCGCTGAAGAGGTACTGG-3'.

[0058] (2) Design of targeted homologous recombination vector. The 5'-loxP site insertion region is within intron 2. The upstream homology arm length is 1626 bp. The homology arm sequence is shown in Figure 5 The 3'-loxP site insertion region is in intron 3, and the downstream homology arm sequence is 725 bp. Figure 6. The size of the effective CKO region after the homologous recombination vector is 2253bp. Cas9 nuclease (Cat. No.: #M0386M) was purchased from NEB (Beijing) Co., Ltd. It is an RNA-mediated nuclease that can catalyze the cutting of specific sites of double-stranded DNA. The gRNA combination, Cas9 nuclease and homologous recombination vector were co-injected into the fertilized egg to insert two Lox-P sites in the same direction into both ends of exon 3 of the MYH11 gene to prepare flox mice.

[0059] 2. Knockout mouse strain establishment process

[0060] The chimeric offspring F0 (Founder) obtained after pronuclear injection was mated with wild-type mice to produce F1 generation heterozygous mice. After F1 was hybridized with Vasa-Cre mice, F2 generation flox heterozygous mice (Flox / +, Vasa-Cre) were obtained. The overall process of knockout mouse establishment is shown in Figure 7 Flox mice need to first verify the accuracy of the homology arm sequence and whether the loxP sites have been inserted into both ends of exon 3. All primer design strategies are shown in Figure 8 Primers F1 and R1 were designed, and the PCR region was the upstream homology arm, 5'-loxP site, exon 3 and 3'-loxP site. The R1 primer sequence included the 3'-loxP sequence. The primer information is as follows (SEQ ID NO4-5):

[0061] F1:5'-AGGAGCATCGGGTCCTCATTTAT-3',

[0062] R1:5'-GTGGATTCGGACCAGTCTGA-3'.

[0063] The size of the PCR product is about 4 kd, Tm = 60 ° C. The electrophoresis results of the PCR product are shown in Fig. 9 ; Among them, lanes 1, 4, 8 and 21 are the results of PCR using the genome of F4 generation flox mice as template, lane M is the molecular marker, lane WT is the control group wild-type mice, and lane Water is the PCR negative control group using sterile water as template.

[0064] Primers F2 and R2 were designed, and the PCR region was 5'-loxP site, exon 3 and 3'-loxP site and downstream homology arm. The F2 primer sequence included the 5'-loxP sequence. The primer information is as follows (SEQ ID NO6-7):

[0065] F2:5'-ACGTAAACGGCCACAAGTTC-3',

[0066] R2:5'-CGTGAAATGGGCATAGTCAAACT-3'.

[0067] The size of the PCR product is about 3.4 kd, Tm = 60 ° C. The electrophoresis results of the PCR products are shown in Figure 10; wherein, lanes 1, 2, 8 and 21 are PCR products using the genome of the F4 generation flox mice as a template, lane M is a molecular marker, lane WT is a control group of wild-type mice, and lane Water is a PCR negative control group using sterile water as a template.

[0068] Primers F3 and R3 were designed, and the PCR region was used for sequencing identification to detect the sequence accuracy of the loxP site and CKO region. The primer information is as follows (SEQ ID NO8-9):

[0069] F3:5'-TTTCAGCGGCATCTCTAGTTGT-3'

[0070] R3:5'-TTGCTCATTCACAGTCACATAGCC-3'

[0071] The genome of the progeny F2 flox mouse sequence 1 was used as a template, and the PCR product was connected with the pMD19-T vector (purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd., catalog number: 6013) and sent to Sangon Biotechnology (Shanghai) Co., Ltd. for sequencing. The sequencing results are shown in Fig.11 The results showed that the loxP site had been successfully inserted and the CKO sequence had not been mutated due to the gene editing process.

[0072] flox mice and testis-specific Vasa-cre tool mice (purchased from Saiye Biotechnology Co., Ltd., strain name: C57BL / 6J-Ddx4 em1cyagen Before the flox mice were hybridized with mice expressing Cre recombinase, the MYH11 gene was expressed normally; when the flox mice were hybridized with Vasa-cre mice, the MYH11 gene was knocked out in the testicular tissue, while the gene was expressed normally in other tissues.

[0073] 3. Genotyping of knockout mice

[0074] For mice bred in the F3 generation and later, primers were designed according to the positions shown in the diagram to identify the CKO region. Two pairs of primers were designed. The PCR product corresponding to the first pair of primers was used to identify whether the mice were homozygous or heterozygous, and the PCR product corresponding to the second pair of primers was used to identify whether the mice were transferred with the Vasa-cre gene. See the primer design strategy for details. Fig.12 .

[0075] The first pair of PCR primer sequences (Annealing Temperature 60.0°C) (SEQ ID NOs 10-11):

[0076] F5:5'-TAGAAAGTTCCCATGACTTTTCCCA-3',

[0077] R5:5'-GTCCCTTCAGAAAGAACTAACGTG-3'.

[0078] The PCR product of homozygous mice is 200bp, the PCR product of heterozygous mice is 200bp and 138bp, and the PCR product of wild-type mice is 138bp. In this example, the genome of CKO heterozygous mice (Flox / +, Vasa-Cre) was used as a template for PCR. The electrophoresis diagram is shown in Fig.13 Among them, lane 15 is the PCR electrophoresis result of CKO heterozygous mice, lane M is the molecular marker, lane WT is the control group wild-type mice, and lane Water is the PCR negative control group using sterile water as the template.

[0079] Design the second pair of PCR primer sequences (Annealing Temperature 60.0°C) (SEQ ID NO12-13):

[0080] Vasa-MF:5'-CACGTGCAGCCGTTTAAGCCGCGT-3'

[0081] Vasa-MR:5'-TTCCCATTCTAAACAACACCCTGAA-3

[0082] The PCR product of Cre homozygous mice and Cre heterozygous mice is 240bp, and the PCR product of wild-type mice is 0. In this example, the genome of heterozygous CKO heterozygous mice (Flox / +, Vasa-Cre) was used as a template for PCR. The electrophoresis diagram is shown in Fig.14 Among them, lane 15 is the PCR electrophoresis result of CKO heterozygous mice, lane M is the molecular marker, lane WT is the control group wild-type mice, and lane Water is the PCR negative control group using sterile water as the template.

[0083] Four CKO heterozygous mice (Flox / +, Vasa-Cre) obtained from the F4 generation were taken, and DNA was extracted from the tail tip tissue for Southern Blot experiments to detect whether the homologous recombination vector was integrated into the exon 3 of the MYH11 gene, and to verify whether the Cas9 targeting system had off-target effects. The 5' probe and 3' probe were designed, and the primer sequences were as follows (SEQ ID NOs 14-17):

[0084] 5'Probe forward primer:5'-AAAGAGGAGATGGAAATGGGTC TGG-3',

[0085] 5'Probe reverse primer:5'-TGAGCCCTGACCTTGCTACCTTCT -3'.

[0086] 3'Probe forward primer:5'-ACACACCCTGCCTGCTGTTGTAG TTT-3',

[0087] 3'Probe reverse primer:5'-GGCCAAGACCCAGTTGTGTCTCTCTT- 3'.

[0088] DNA endonucleases Avrll and AflII were selected to perform restriction endonuclease on the CKO region of the MYH11 gene in the F1 generation after homologous recombination. The overall detection strategy is shown in Fig.15 ; Among them, the 5'Probe-AvrII probe can capture the 7.64kb WT fragment and the 4.17kb MT fragment; the 3'Probe-AflII probe can capture the 4.77kb WT fragment and the 3.38 kb MT fragment. The Southern Blot detection, after the 5'probe and 3'probe capture, the analysis exposure results are shown in Fig.16 The results showed that the heterozygous loxP site was successfully integrated in 4 CKO mice in the F1 generation, and the off-target efficiency of the Cas9 targeting system was 0.

[0089] 4. Analysis of male reproductive system development in knockout mice

[0090] The CKO heterozygous male mice of the F4 generation obtained by genotyping were anesthetized and sperm from the epididymis were collected for sperm motility analysis. The results of the comparative analysis with those of wild-type mice are shown in Fig.17 ; Among them, the sperm motility and sperm vitality of CKO heterozygous mice were slightly lower than those of wild-type mice, but the difference was not statistically significant, while the sperm linear velocity and curve velocity were significantly decreased, and the difference was statistically significant; the sperm deformity rate, lateral swing amplitude, swing, whipping frequency, linearity, movement angle and progressiveness of CKO heterozygous mice were no different from those of wild-type mice.

[0091] HE staining was performed on the testis tissues of CKO heterozygous male mice and wild-type mice. Fig.18Among them, the testicular tissue structure of wild-type mice (Myh11+ / +) was normal, the seminiferous epithelium and basement membrane were intact, the seminiferous tubules were closely arranged, the morphology was clear, the boundaries were complete, the upgraded cells at all levels were clearly layered, and the interstitial cells were evenly arranged. The testicular tissue of CKO heterozygous mice (Myh11+ / -) had incomplete boundaries of seminiferous tubules (arrows in the figure), the seminiferous epithelium was separated from the basement membrane (triangle in the figure), the lumen of the seminiferous tubules was narrow, and the number of primitive spermatogenic cells increased.

[0092] The bladder tissues of CKO heterozygous male mice and wild-type mice were stained with HE. Fig.19 Among them, the bladder structure of wild-type mice (Myh11+ / +) is regular, the mucosal folds are intact, and the smooth muscles are neatly arranged. The bladder tissue volume of CKO heterozygous mice (Myh11+ / -) is significantly increased, the bladder wall is significantly thinner, the mucosal barrier is thinner and partially shed, the number of smooth muscle layers increases, the arrangement is irregular, and the smooth muscle gaps increase (arrows in the figure).

[0093] HE staining was performed on lung tissue, thoracic aorta, muscle tissue and muscle tissue of CKO heterozygous mice and wild-type mice. The results are shown in Fig. 20 . Among them, the HE staining results of lung tissue showed that the airway lumen of wild-type mice lung tissue was smooth, the structure was clear, and there was no hyperplasia of the alveolar septum; the airway lumen of CKO heterozygous mice lung tissue was intact, and the alveolar size and alveolar septum did not change significantly compared with the wild type. The HE staining results of the thoracic aorta showed that the inner, middle and outer three-layer wall structure of the wild-type mouse thoracic aorta was intact, the wall thickness was uniform, and the vascular smooth muscle cells were clearly visible and arranged neatly; the thoracic aorta of CKO heterozygous mice was intact, and the smooth muscle was arranged neatly, with no significant difference compared with wild-type mice. The HE staining results of muscle tissue showed that the muscle bundle structure of wild-type mice and CKO heterozygous mice was dense and smooth, and the muscle bundles were arranged regularly, with no significant difference between the two.

[0094] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention. Sequence Listing <110> Inner Mongolia Medical University <120> A method for constructing a testicular MYH11 gene knockout mouse model and its application <130> KHP211124371.2 <160> 17 <170> SIPOSequenceListing 1.0 <210> 1 <211> 2253 <212> DNA <213> Artificial Sequence <400> 1 taaggttagg acccctgctg tgcacacccc actccctcct cctcaggagg gagttacata 60 tgtcattgct gcttttgttg caaattaaaa actataaaat taaaacaata acattgttat 120 tggtgtcctt ttggtttatg ttgttttttt tgagacagtt tctctgtgaa gccctggctg 180 tccttgaact catgctgtag acaacttggt gtgaactcag agatctgttt gcctctgcct 240 ccgagtgctg gaattaaagg tgtgcaccac cactgcccat tggtattctt tttttttttt 300 tttttttttt ttttttttgt tattcgagac agagcttctc tttatagctc tagctgtcca 360 ggtcagcttt gtagaccagg ctggcctcga actcagaaat ccaactgcct ctgcctcccg 420 agtgctggga ttaaaggcgt gcaccaccat gcctggctgc ccattggtat tctttaagta 480 gcatcctatc ttttaatcaa ataaaaataa aaatgagatc aataaaaacg gaattttggg 540 agagctgggg agatggctca gttgctaagg tgcttgctgc tcaagcatga gaacctgagg 600 <h2 style=";text-align:left;direction:ltr">tgagttccca gcccccatgg ggaaaggctg ggtgtagtga gccaagctct tgtcatctga 660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgctggggga ggaggagaat aggggacccg tggagttgat gcttacccag tctagtcaga 720<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttggtgagtt ctaggttcag tgagggagcc catcctaaat aataaagagt agggactgga 780<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gaggacaccc catgctgacc cctgttctcc acatgtctgc acacatgtgc atgtgtaccc 840<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tacatatgaa catgtgcaca gagacttaag ttgtgtcccc gaggcttagt gaggtagagg 900<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> acagaggtgg gttgggagct tagaagtgag tccagagctg gctaagtcct aaccacaggt 960<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgtctgggaa 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aagcaaaaag aatgtcggtc tgcagcaagg 1440 aggggcagg gcactcaggg cttgagcaaa tacgggcatc gggaatcgcc ctctctggct 1500 tctgccatcc ctgtggctgt tccagagccc tggcccagct agcagctaga gagaaaaaaa 1560 tgggtatggg catggatgag ctgcagagac ctgggttgcc atctgccagt ttccatccct 1620 ggaatcacgt tcaggtatct ttttcttcct aacctcagtt ttcttgtatg tgaaagggga 1680 taatagtctc tatgttttca catggagggc ctttgcacac acactccaag tccaggtgtg 1740 gcagcttgtg gattctttat ctgaaaatgt tgagaccaga gccattgcag atttggaact 1800 gacctcccac ccccagattt aggaatattt ccatatacgc atgatgagca tttgtggggc 1860 tgaaactcaa gtgtaaaaat aacatttatt tacatctttt ctttatttt tatagttctg 1920 aggtaatttt ggacaatatt tcaaacaatt ttgttcatga agcaatgttt tgtcaagtgg 1980 aattttccca ttgtggttc acgtttggac taaaaaagt ttcagatttt ggattttgg 2040 gtttgggctt ctcagcccat gtgactgcca tcttgaatga gcatttctat tgggtcaaaa 2100 tggcagatac atcacaaaat aatagggaaa gtgttaattt aagagtcgca ctgaaaataa 2160 tttttatacc tgcctataat agaataatta ctattgagat aaagatatga atgaagtatt 2220 ctagaaagtt cccatgactt ttcccagcca gta 2253 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <400> 2 caggggtcct aaccttatat agg 23 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <400> 3 tgtgtgcgct gaagaggtac tgg 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <400> 4 aggagcatcg ggtcctcatt tat 23 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 gtggattcgg accagtctga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 acgtaaacgg ccacaagttc 20 <210> 7 <211> twenty three <212> DNA <213> Artificial Sequence <400> 7 cgtgaaatgg gcatagtcaa act 23 <210> 8 <211> twenty three <212> DNA <213> Artificial Sequence <400> 8 tttcagcggc atctctagtt tgt 23 <210> 9 <211> twenty four <212> DNA <213> Artificial Sequence <400> 9 ttgctcattc acagtcacat agcc 24 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <400> 10 tagaaagttc ccatgacttt tccca 25 <210> 11 <211> twenty four <212> DNA <213> Artificial Sequence <400> 11 gtcccttcag aaagaactaa cgtg 24 <210> 12 <211> twenty four <212> DNA <213> Artificial Sequence <400> 12 cacgtgcagc cgtttaagcc gcgt 24 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <400> 13 ttcccattct aaacaacacctgaa 25 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <400> 14 aaagaggaga tggaaatggg tctgg 25 <210> 15 <211> twenty four <212> DNA <213> Artificial Sequence <400> 15 tgagccctga ccttgctacc ttct 24 <210> 16 <211> 26 <212> DNA <213> Artificial Sequence <400> 16 acacaccctg cctgctgttg tagttt 26 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <400> 17 ggccaagacc cagttgtgtc tctctt 26

Claims

1. A method for constructing a testicular MYH11 gene knockout mouse model, characterized in that: include: A targeted homologous recombination vector is constructed, and the targeted homologous recombination vector, gRNA and Cas9 protein are introduced into a fertilized mouse egg. The developed mice are hybridized with wild-type mice to obtain F1 generation heterozygous mice; the F1 generation heterozygous mice are hybridized with mice that specifically express Vasa-Cre enzyme in testicular tissue to obtain F2 generation mice, and a testicular MYH11 gene knockout mouse model is obtained after identification; The 5'-loxP site insertion region in the targeted homologous recombination vector is located in intron 2, and the 3'-loxP site insertion region is located in intron 3; The targeting region of the targeted homologous recombination vector includes the nucleotide sequence shown in SEQ ID NO.1; The gRNA comprises: gRNA1:5'-CAGGGGTCCTAACCTTATATAGG-3', gRNA2:5'-TGTGTGCGCTGAAGAGGTACTGG-3'.

2. A primer combination for constructing a testicular MYH11 gene knockout mouse model, characterized in that: include: Upstream homology arm amplification primer pair: F1:5'-AGGAGCATCGGGTCCTCATTTAT-3', R1:5'-GTGGATTCGGACCAGTCTGA-3'; and, Downstream homology arm amplification primer pair: F2:5'-ACGTAAACGGCCACAAGTTC-3', R2: 5'-CGTGAAATGGGCATAGTCAAACT-3'; and Primer pair for loxP site detection: F3:5'-TTTCAGCGGCATCTCTAGTTGT-3', R3:5'-TTGCTCATTCACAGTCACATAGCC-3'.

3. A kit, characterized in that: The kit comprises the primer combination according to claim 2.

4. Use of the primer combination according to claim 2 or the kit according to claim 3 in constructing a MYH11 gene knockout mouse model.

5. Use of the construction method according to claim 1, or the primer combination according to claim 2, or the kit according to claim 3 in constructing a male reproductive defect mouse model.

6. Use of the construction method according to claim 1, or the primer combination according to claim 2, or the kit according to claim 3 in constructing a mouse model of urinary system diseases.

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