sgRNA combination, pig model and construction method for knocking out part of the conserved sequence of the pig RBM20 gene

Abstract

The present invention discloses a sgRNA combination for knocking out a part of the conservative sequence of the pig RBM20 gene, a pig model and a construction method, wherein the sgRNA combination comprises a first sgRNA and a second sgRNA; the sense strand of the DNA sequence of the first sgRNA is shown in SEQ ID No: 1, and the antisense strand is shown in SEQ ID No: 2; the sense strand of the DNA sequence of the second sgRNA is shown in SEQ ID No: 21, and the antisense strand is shown in SEQ ID No: 22. The present invention selects sgRNAR6 and sgRNAR10 with high targeting efficiency to achieve efficient knockout of the 9th exon of the RBM20 gene from PFFs, and successfully obtains the 9th exon knockout pig of RBM20 through somatic cell nuclear transplantation and embryo transplantation technology, providing a new animal model and more biological material options for the study of the mechanisms of muscle system diseases or muscle damage, aging, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a sgRNA combination, a pig model and a construction method for knocking out a part of the conserved sequence of the pig RBM20 gene. Background Art

[0002] Skeletal muscle is an important organ for maintaining normal human activities. It plays an important role in movement, supporting breathing, heat production and maintaining homeostasis. Skeletal muscle dysfunction is one of the common clinical diseases. Injury, disease and aging are common causes of its functional defects. How to effectively treat skeletal muscle injury, improve healing quality and promote the injury repair process has become a difficult problem to be solved in clinical research. The repair of adult skeletal muscle injury mainly depends on skeletal muscle satellite cells. Satellite cells are mononuclear cells located between the sarcolemma and the basement membrane. They have strong differentiation potential, and their differentiation ability is the key to the success or failure of muscle tissue regeneration. When skeletal muscle is damaged, satellite cells as stem cells are first activated, proliferated, and differentiated into myoblasts. Myoblasts fuse to form muscle fibers, thereby repairing and regenerating damaged muscle tissue. Therefore, the differentiation potential of satellite cells makes them a potential resource for treating hereditary muscle tissue diseases and repairing damaged skeletal muscle.

[0003] RBM20 is located on the long arm of chromosome 10, contains 14 exons, and encodes a protein containing 1227 amino acids. The protein contains two zinc finger domains, a glutamate-rich region, a leucine-rich region, an RNA binding domain (RNA recognition motif), and an arginine / serine-rich region (RS domain). Studies have shown that RBM20 has the function of regulating the alternative splicing of genes and can regulate the alternative splicing of Titin, an important functional protein in sarcomeric proteins. Studies on a large number of clinical samples of heart-related diseases have shown that mutations in RBM20 can cause human dilated cardiomyopathy. RBM20-deficient rats and mice develop dilated cardiomyopathy, which is highly similar to human dilated cardiomyopathy caused by RBM20 mutations.

[0004] However, although recent studies have linked human RBM20 to dilated cardiomyopathy (DCM), the function, regulation, and substrates of RBM20 remain unclear. In fact, RBM20 is expressed not only in myocardium, but also in skeletal muscle. Therefore, research on the function of RBM20 needs to be more comprehensive and in-depth, and cannot be limited to myocardium. In particular, the role of RBM20 in skeletal muscle is worth exploring, because studies have shown that patients with skeletal muscle diseases may have combined cardiac dysfunction, which suggests that our research on skeletal muscle function may contribute to breakthroughs in the treatment of cardiomyopathy. In addition, due to the high similarity between pigs and humans in anatomy, physiology, genetics, and disease progression, pigs have gradually become an ideal animal model for studying the mechanisms of human diseases in recent years.

[0005] In addition, skeletal muscle dysfunction is one of the common clinical diseases. How to effectively treat skeletal muscle system diseases, delay muscle aging and atrophy, and promote muscle damage and reconstruction is a very meaningful research direction. However, there are currently few models for skeletal muscle disease and function research. The only commonly used one is the mdx mouse, which is a mouse model for human Duchenne muscular dystrophy (DMD). Therefore, it is particularly necessary to establish other skeletal muscle disease models for skeletal muscle growth and development or the function of pathogenic genes. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to address the problem that there are few models for skeletal muscle disease and function research in the prior art, thereby providing a sgRNA combination, pig model and construction method for knocking out part of the conserved sequence of the pig RBM20 gene, which can provide new animal models and more biomaterial selection directions for the study of muscle system diseases or muscle damage, aging and other mechanisms.

[0007] In order to achieve the above object, the present invention provides a sgRNA combination for knocking out a part of the conservative sequence of the pig RBM20 gene, wherein the sgRNA combination comprises a first sgRNA and a second sgRNA; and,

[0008] The sense strand of the DNA sequence expressing the first sgRNA is shown in SEQ ID No: 1, and the antisense strand is shown in SEQ ID No: 2;

[0009] The positive strand of the DNA sequence expressing the second sgRNA is shown as SEQ ID No: 21, and the antisense strand is shown as SEQ ID No: 22.

[0010] Preferably, the sgRNA combination action site is located on the 9th exon of the pig RBM20 gene.

[0011] The present invention also provides a targeting plasmid combination for knocking out a part of the conservative sequence of the pig RBM20 gene, wherein the targeting plasmid combination is obtained by connecting the double-stranded DNA of the first sgRNA and the second sgRNA described above to a vector.

[0012] Preferably, the vector is selected from pX330 vector.

[0013] The present invention also provides a porcine fetal fibroblast gene knockout cell, which is obtained by transfecting the above-mentioned targeting plasmid combination into porcine fetal fibroblasts, culturing them, and screening them.

[0014] The present invention also provides a method for constructing a functionally defective pig model, which comprises: using the pig fetal fibroblast gene knockout cells as donor cells, and obtaining the functionally defective pig model by somatic cell nuclear transplantation.

[0015] The present invention also provides a functionally defective pig model, which is obtained by using the construction method described above.

[0016] Preferably, the functional defect pig model is a skeletal muscle functional defect pig model.

[0017] Through the above technical scheme, the present invention selects two sgRNAs, sgRNA R6 and sgRNA R10, with high targeting efficiency to achieve efficient knockout of the 9th exon of the RBM20 gene from PFFs. Then, RBM20 exon 9 knockout pigs were successfully obtained through somatic cell nuclear transplantation and embryo transplantation technology. After testing, it was found that pigs with exon 9 deletion of the RBM20 gene all showed skeletal muscle growth retardation, and the proliferation rate and differentiation ability of their satellite cells were also severely inhibited. The proliferation and differentiation ability of satellite cells represent the potential for skeletal muscle reconstruction. It is confirmed that the RBM20 exon 9 knockout pig constructed by the present invention can be used as an animal model of skeletal muscle function defects, providing a new animal model and more biomaterial options for the study of muscle system diseases or muscle damage, aging and other mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0019] Figure 1A This is a diagram showing the result of the digestion of the amplified product in Example 1 after digestion with T7E1;

[0020] Figure 1B is a diagram of the Sanger sequencing results of sgRNAR6 and sgRNAR10 in Example 1;

[0021] Figure 1C It is a schematic diagram of the strategy for sgRNA target site design and RBM20E9D identification in Example 1;

[0022] Figure 1D is a diagram showing the identification result of the amplified product of RBM20E9D after amplification with R9F / R9R primers in Example 1;

[0023] Figure 1E This is a diagram showing the molecular level identification results of three RBM20E9D piglets in Example 2;

[0024] Figure 2AIt is a picture of the H&E staining result of the cross-sectional and longitudinal section morphology of muscle fibers of the muscle tissue of WT (wild-type pig) and RBM20E9D pig in detection example 1;

[0025] Figure 2B is a statistical analysis result diagram of the total cross-sectional area of ​​muscle fibers in muscle tissue of WT (wild-type pig) and RBM20E9D pig in detection example 1 (n=3, *P<0.05);

[0026] Figure 2C is a schematic diagram of LAMININ staining to mark the morphology of muscle fibers in detection example 1, wherein LAMININ is used to mark the muscle fiber structure, and DAPI is used to mark the cell nucleus, and the scale bar = 50 μm;

[0027] Figure 3A This is the result of measuring the proliferation of RBM20E9D satellite cells by CCK-8 in detection example 2 (n=3);

[0028] Figure 3B is the result of evaluating the proliferation of RBM20E9D satellite cells by EdU staining in detection example 2, wherein the scale bar = 100 μm;

[0029] Figure 3C is the statistical result of EdU staining in detection example 2 (n=3), **P<0.01;

[0030] Figure 4A It is the crystal violet staining image of satellite cells in the control group and RBM20E9D group after 3 days of differentiation in detection example 3, scale bar = 200 μm;

[0031] Figure 4B is the MyHC immunofluorescence staining image of satellite cells in the control group and RBM20E9D group after 3 days of differentiation in detection example 3, scale bar = 200 μm;

[0032] Figure 4C is a statistical analysis result diagram of the differentiation index of satellite cells in the control group and RBM20E9D group after differentiation in detection example 3 (the number of nuclei in myotubes containing 2 or more nuclei / the total number of nuclei, n=3);

[0033] Figure 4D is a statistical analysis result diagram of the average number of nuclei in MyHC-positive cells of the satellite cells of the control group and RBM20E9D group after differentiation in detection example 3 (n=3);

[0034] Figure 4E is the immunofluorescence staining of α-actin in satellite cells of the control group and RBM20E9D group after 3 days of differentiation in detection example 3, scale bar = 50 μm;

[0035] Figure 4FThis is a graph showing the qRT-PCR detection results of the differentiation marker genes MyoD and MyoG in the control group and the RBM20E9D group 2 days after induction of differentiation in Example 3 (n=3), **P<0.01. DETAILED DESCRIPTION

[0036] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0037] Specifically, the present invention provides a skeletal muscle function defect pig model (i.e., RBM20 gene exon 9 knockout pig model, denoted as RBM20E9D) and a construction method thereof, providing a new animal model for the study of the function of RBM20 in skeletal muscle and the study of the mechanism of muscle system diseases or muscle damage, aging, etc. The following is further described in detail in conjunction with specific embodiments.

[0038] In the present invention, the pig breed used is a miniature pig purchased from a pig breeding farm in Zhuozhou, Hebei Province. The porcine fetal fibroblasts (denoted as PFFs) used are isolated from 30-day-old miniature pig fetuses (separation is achieved by conventional means that can be understood and operated by those skilled in the art. In the present invention, the method described in "Yu Yongsheng, Cao Yang, Luo Xiaotong, et al. Isolation and gene transfection of porcine fetal fibroblasts. Jilin Agricultural Science, 2014, 39(04): 50-53" is used for operation). Plasmid pX330 is a conventional commercial product with a brand number of #42230 from Addgene, pGK (Neo) plasmid is a gift from China Agricultural University (specifically, the plasmid disclosed and used in the article "doi: 10.7150 / ijbs.34269"), DMEM / F12, horse serum and DMEM culture medium are conventional commercial products from Gibco, bFGF is a conventional commercial product from PeproTech, Matrigel is a conventional commercial product from BD Biosciences,

[0039] Amaxa Basic NucleofectorKit is a conventional commercial product of Lonza with the product number VPI-1002.

[0040] Example 1, obtaining RBM20 exon 9 knockout pig fetal fibroblasts (RBM20E9D):

[0041] According to the sequence of exon 9 of RBM20 (GenBank accession number: NM_214173.1) in PFFs, eight sgRNAs (denoted as R1, R2, R3, R4, R5, R6, R7 and R11) were designed upstream and three sgRNAs (denoted as R8, R9 and R10) were designed downstream. The double-stranded DNA sequences corresponding to R1-R11 are shown in Table 1. Specifically, R1 corresponds to SEQ ID No: 5 and SEQ ID No: 6, R2 corresponds to SEQ ID No: 7 and SEQ ID No: 8, R3 corresponds to SEQ ID No: 9 and SEQ ID No: 10, R4 corresponds to SEQ ID No: 11 and SEQ ID No: 12, R5 corresponds to SEQ ID No: 13 and SEQ ID No: 14, R6 corresponds to SEQ ID No: 1 and SEQ ID No: 2, R7 corresponds to SEQ ID No: 15 and SEQ ID No: 16, R11 corresponds to SEQ ID No: 3 and SEQ ID No: 4, R8 corresponds to SEQ ID No: 17 and SEQ ID No: 18, R9 corresponds to SEQ ID No: 19 and SEQ ID No: 20, and R10 corresponds to SEQ ID No: 21 and SEQ ID No: 22.

[0042] The double-stranded DNA corresponding to each of the above 11 sgRNAs was connected to the plasmid pX330 to obtain 11 corresponding targeting plasmids (respectively recorded as pX330-R1, pX330-R2, pX330-R3, pX330-R4, pX330-R5, pX330-R6, pX330-R7, pX330-R8, pX330-R9, pX330-R10, and pX330-R11).

[0043] 4 μg of each of the 11 targeting plasmids obtained above were taken and transferred into PFFs through the N024 electroporation procedure. After 48 hours, the cells were collected and the genomes were extracted to obtain 11 corresponding target fragments, which were amplified, purified and recovered. Specifically, the target fragments obtained corresponding to R1-R7 and R11 were amplified using forward primer 1F (SEQ ID No: 23: 5'-TTGAGCAAGTCAGAGCTCTGAGG-3') and reverse primer 2R (SEQ ID No: 24: 5'-TCCAGGCGCTCATCTAACTCAG-3'), and an amplified product with a length of 1085 bp was amplified; the target fragments obtained corresponding to R8-R10 were amplified using forward primer 3F (SEQ ID No: 25: 5'-TGGGAGGTCCAGGAGGAAAGAC-3') and reverse primer 1R (SEQ ID No: 26: 5'-TCACAGTCATCAGCTCCTGGGTC-3'), and an amplified product with a length of 830 bp was amplified. The obtained amplified product was digested with T7E1 and subjected to 2.5% agarose gel electrophoresis. The theoretical cutting band size of the T7E1 digested fragment is shown in Table 1.

[0044] The results of the digestion of the amplified product by T7E1 are as follows Figure 1A As shown, it reflects the targeting efficiency of the RBM20 gene digested by T7EI. Figure 1A It can be seen that the upstream targeting site sgRNAR6 and the downstream targeting site sgRNAR10 of exon 9 were effectively cut, and the cut size was in line with expectations. The amplified products were connected to the TA clone and Sanger sequencing was performed to determine the targeting efficiency of sgRNA R6 and sgRNA R10. According to the sequencing results, the targeting efficiency of sgRNA R6 was calculated to be 27.2%, and the targeting efficiency of sgRNA R10 was 20.0% (targeting efficiency = number of mutant clones / total number of clones) (specific results are shown in Figure 1B As shown, Figure 1B The red box in the middle indicates the PAM sequence position and the targeting sequence). Based on this, the two sgRNAs R6 and R10 were selected for the subsequent knockout of the 9th exon of RBM20.

[0045] The vector was electrotransfected according to the protocol provided by Amaxa Basic NucleofectorKit (Lonza, VPI-1002). Specifically, 2 μg of px330-sgRNAR6, 2 μg of px330-sgRNAR10 (i.e., the total amount of targeting plasmid was 4 μg) and 1 μg of pGK (Neo) plasmid were added to 150 μL of electrotransfection solution. The above plasmid-electrotransfection solution mixture was resuspended in about 1×10 6PFFs digested with trypsin were mixed and transferred to Lonza electroporation cups and electroporated using Lonza electroporator using N024 electroporation program. The electroporated cells were added to 10-15 10 cm 2 Petri dishes (10 cm each 2 Add 10 4 cells), and screened for 9-12 days with DMEM medium containing G418 (final concentration 600 μg / mL, Promega). Resistant clones were picked up with cloning columns for subculture to obtain RBM20E9D PFFs monoclones (hereinafter referred to as RBM20E9D PFFs). 30% of the subcultured cells from each clone were used for genomic DNA extraction. The genotype of the cloned fibroblasts was identified using the following primers: forward primer R9F (SEQ ID No: 27: 5'-TTGAGCAAGTCAGAGCTCTGAGG-3') and reverse primer R9R (SEQ ID No: 28: 5'-TCACAGTCATCAGCTCCTGGGTC-3'). The amplification product of wild-type PFFs was 2068 bp, and the amplification product of RBM20E9D PFFs was 1107 bp. The schematic diagram of the identification results of the sgRNA target site and the amplification product of RBM20E9D is shown in Figure 1C As shown, through Figure 1C It can be seen that after the simultaneous action of sgRNA R6 and sgRNAR10, the amplified product of wild-type porcine fetal fibroblasts is 2068 bp, and the amplified product of porcine fetal fibroblasts after RBM20 exon 9 knockout is 1107 bp. The identification results of the product size after amplification with R9F / R9R primers are as follows Figure 1D As shown, the red arrow indicates the RBM20E9D homozygous PFFs clone. Among the 98 G418-resistant PFFs clones, 5 were identified as RBM20 exon 9 knockout (respectively 11#, 14#, 47#, 61# and 85#), and were homozygous clones.

[0046] Table 1

[0047]

[0048]

[0049]

[0050] Example 2: Production of RBM20E9D pigs by somatic cell nuclear transplantation:

[0051] The homozygous RBM20 exon 9 deletion PFFs (specifically selected here) Figure 1DRBM20E9D (numbered 85# shown in the figure) was digested with trypsin and kept at 4°C as SCNT (somatic cell nuclear transfer) donor cells. The process of somatic cell nuclear transplantation (SCNT) is as follows: the oocytes used for SCNT (ovaries purchased from a slaughterhouse) were cultured in vitro for 40-44 hours, and then the donor RBM20E9D cells were transferred to the perivitelline space between the cytoplasm and cytoplasm of the enucleated oocytes. The reconstructed oocytes were cultured overnight at 38.5°C in porcine fertilized egg culture medium, and 1453 reconstructed embryos were obtained by SCNT. The blastocysts were transferred to the oviduct umbrella of estrus recipient sows (specifically 5 surrogate sows). 28 days after transplantation, an ultrasound examination confirmed that one surrogate sow was pregnant and successfully gave birth to 3 piglets. The ear skin of newborn piglets was collected and genomic DNA was extracted for genotyping. The molecular level identification results of three piglets (numbered 114001#, 114002# and 114003#) are as follows: Figure 1E As shown, the extracted genomic DNA was amplified using the forward primer R9 shown in SEQ ID No: 27 and the reverse primer R9R shown in SEQ ID No: 28 and then identified. It can be seen that the 9th exon of the RBM20 gene of the three piglets was knocked out.

[0052] Example 3: Isolation, culture and differentiation induction of porcine skeletal muscle satellite cells:

[0053] Cells were extracted from the skeletal muscle of the piglet (i.e., RBM20E9D pig) in Example 2. The muscle tissue was cut and minced into minced meat using sterile scissors; and digested with a digestion working solution at 37°C for 1 h. The digestion working solution contained a final concentration of 2 mg·mL -1 Dispase II and 4 mg mL -1 Collagenase II high glucose DMEM medium solution, according to 1cm 3 The muscle tissue requires 5 to 10 mL of digestion working solution for digestion. The digestion solution was filtered through 100 μm and 40 μm cell sieves in turn. The erythrocytes were broken with 3 mL of erythrocyte lysis solution. Satellite cells were cultured with complete skeletal muscle satellite cell growth medium at 37°C and 5% CO2. The complete growth medium was DMEM / F12 medium solution (Gibco), supplemented with 20% FBS (Foundation), 1% penicillin-streptomycin mixture, and 5 ng mL -1 bFGF (PeproTech). The culture dishes used for culture were treated with Matrigel (BD Biosciences). To induce differentiation, cells were incubated in DMEM medium (DMEM, Gibco) containing 2% horse serum (HS; Gibco) and 1% penicillin-streptomycin.

[0054] Test example 1: Detection of skeletal muscle growth and development in pigs with RBM20 exon 9 knockout:

[0055] The longissimus dorsi muscle of the 7-day-old piglets (i.e., RBM20E9D pigs) and wild-type pigs (i.e., untreated miniature pigs) obtained in Example 2 was tested. The H&E staining results showed that ( Figure 2A and Figure 2B As shown, Figure 2A Scale bar = 200 μm). Compared with the skeletal muscle of wild-type pigs, the skeletal muscle fiber bundles of RBM20E9D pigs were small, the cross-sectional area of ​​the muscle fiber bundles was significantly smaller than that of wild-type individuals, and the muscle fibers were loosely arranged, with large gaps and irregular arrangement. The results of immunofluorescence staining with LAMININ antibody were consistent with those of H&E staining ( Figure 2C The results also show that the muscle tissue of RBM20E9D pigs is small, which indicates that the knockout of exon 9 of RBM20 leads to delayed muscle development.

[0056] Test example 2: Detection of proliferation of skeletal muscle satellite cells in pigs with RBM20 exon 9 knockout:

[0057] The skeletal muscle satellite cells isolated in Example 3 were purified and cultured to form the RBM20E9D satellite cell line. The proliferation of the cells was detected using a CCK-8 detection kit, and it was found that compared with the control group, the knockout of RBM20 exon 9 resulted in a significant decrease in the proliferation rate of satellite cells ( Figure 3A This result was also confirmed by labeling proliferating cells with EdU ( Figure 3B and Figure 3C As shown, P<0.05).

[0058] Test example 3: Detection of differentiation ability of pig skeletal muscle satellite cells knocked out in exon 9 of RBM20:

[0059] RBM20E9D satellite cells (isolated in the manner of Example 3) and a control group (operated in the manner of Example 3, except that the piglets used were untreated miniature pigs) were induced to differentiate, and cell samples were collected 3 days later for crystal violet staining. The results showed that the deletion of exon 9 of RBM20 inhibited the differentiation ability of satellite cells, and the number of myotubes formed was significantly reduced ( Figure 4A Myosin heavy chain MyHC is a constitutive protein of muscle fibers. Using MyHC antibody to mark mature myotubes, immunofluorescence staining results showed that the deletion of RBM20 exon 9 significantly reduced the number of satellite cells that differentiated into mature myotubes ( Figure 4B). Statistics showed that the differentiation index of RBM20E9D cells (the number of nuclei in myotubes containing two or more nuclei / the total number of nuclei) was significantly lower than that of wild-type cells (31.3% vs 57.4%, P<0.01) ( Figure 4C The average number of nuclei in MyHC-positive cells in the RBM20E9D cell group was also significantly lower than that in the wild-type cell group ( Figure 4D , P<0.01). In general, compared with the control group, the RBM20E9D cell group formed more small myotubes with 2 to 4 nuclei, and few mature large myotubes, and the levels of MYOD and MYOG mRNA were significantly downregulated ( Figure 4F , P<0.01), indicating that the deletion of exon 9 of RBM20 resulted in the impairment of satellite cell differentiation ability. In addition, immunostaining of α-Actinin showed ( Figure 4E ), RBM20 exon 9 deletion leads to disordered satellite cell sarcomere structure, which may further lead to defective muscle fiber function.

[0060] The present invention provides two sgRNAs, sgRNA R6 (located upstream of the 9th exon of RBM20, with a targeting efficiency of 27.2%) and sgRNAR10 (located downstream of the 9th exon of RBM20, with a targeting efficiency of 20.0%), with high targeting efficiency, to achieve efficient knockout of the 9th exon of the RBM20 gene from PFFs. RBM20 exon 9 knockout pigs were then successfully obtained by somatic cell nuclear transplantation and embryo transplantation techniques. It was found that pigs with exon 9 deletion of the RBM20 gene all showed skeletal muscle growth retardation, and the proliferation rate and differentiation ability of their satellite cells were also severely inhibited. The proliferation and differentiation ability of satellite cells represent the potential for skeletal muscle reconstruction. Therefore, RBM20 exon 9 knockout pigs can be used as an animal model for skeletal muscle functional defects, which provides a new animal model and more biomaterial options for the study of muscle system diseases or muscle damage, aging and other mechanisms.

[0061] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0062] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0063] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A sgRNA combination for knocking out a part of the conservative sequence of the pig RBM20 gene, characterized in that: The sgRNA combination includes a first sgRNA and a second sgRNA; and, The sense strand of the DNA sequence expressing the first sgRNA is shown in SEQ ID No: 1, and the antisense strand is shown in SEQ ID No: 2; The sense strand of the DNA sequence expressing the second sgRNA is shown in SEQ ID No: 21, and the antisense strand is shown in SEQ ID No: 22; The sgRNA combination is used to knock out exon 9 of the RBM20 gene.

2. A targeting plasmid combination for knocking out part of the conservative sequence of the pig RBM20 gene, characterized in that: The targeting plasmid combination is prepared by combining the first sgRNA and the second sgRNA of claim 1 The double-stranded DNA was connected to the vector to obtain.

3. The targeting plasmid combination according to claim 2, characterized in that: The vector is selected from pX330 vector.

4. A porcine fetal fibroblast gene knockout cell, characterized in that: The porcine fetal fibroblast gene knockout cells are obtained by transfecting the targeting plasmid combination as described in claim 2 or 3 into porcine fetal fibroblasts, culturing them, and then screening them.

5. A method for constructing a functional defect pig model, characterized in that: The construction method comprises: using the porcine fetal fibroblast gene knockout cells as claimed in claim 4 as donor cells, and obtaining a functional defective pig model by somatic cell nuclear transplantation.

6. The construction method according to claim 5, characterized in that: The functional defect pig model is a skeletal muscle functional defect pig model.

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