CN117587069A - Universal lysosomal storage disease musculoskeletal lesion animal model and construction method - Google Patents
Universal lysosomal storage disease musculoskeletal lesion animal model and construction method Download PDFInfo
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Abstract
The invention provides a general lysosomal storage disease musculoskeletal lesion animal model and a construction method thereof, which comprises the following steps of firstly constructingChmp5 fl/fl Mice, reuse the saidChmp5 fl/fl The mouse is hybridized with the Cre mouse with the specificity of the osteoblast lineage cells, and the universal lysosomal storage disease musculoskeletal lesion animal model is constructed and obtained, so that the CHMP5 gene is conditionally knocked out in the osteoblast related cells, and the method can be used for researching the common pathogenesis of the musculoskeletal related lesions of the lysosomal storage disease and can also be used for screening, identifying and evaluating universal medicines for the diseases. The lysosomal storage disease musculoskeletal lesion animal model constructed by the method is stable, the incidence rate is 100%, the musculoskeletal symptoms of the mice are early onset, and the lesions are typical and comprise extensive periosteum and endostealMembranous hyperplasia, progressive joint mobility decline, and short stature, which more fully mimic musculoskeletal-related lesions of human lysosomal storage disorders.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a general lysosomal storage disease musculoskeletal lesion animal model and a construction method thereof.
Background
Lysosomal storage disorders (Lysosomal storage diseases) are a collective term for a group of about 50 inherited metabolic diseases, resulting from mutations in genes responsible for lysosomal acid hydrolase synthesis, transport and maturation within cells, leading to the inability of biological macromolecules that would otherwise degrade in lysosomes to normally degrade and to storage within lysosomes, thereby affecting the normal functioning of cells, tissues, organs, and being able to cause systemic lesions in the body including the musculoskeletal system.
Musculoskeletal lesions are one of the most common clinical manifestations of lysosomal storage diseases. Currently, most lysosomal diseases still lack effective therapeutic methods, and there is no in-depth understanding of the molecular cellular mechanisms by which these diseases occur. Animal models are key tools for screening and validating effective therapeutic drugs for lysosomal storage diseases and for studying disease pathogenesis. Currently, genetically engineered mice carrying pathogenic genes associated with lysosomal storage disorders have been used for drug validation and evaluation of some subtypes of the disease. However, since lysosomal storage disorders comprise at least 50 different disease types, only a few disease types currently have animal models available, especially a lack of a class of animal models available for studying the common pathogenesis of lysosomal storage disorders.
Disclosure of Invention
In order to solve the problems, the invention provides a general lysosomal storage disease musculoskeletal lesion animal model and a construction method thereof. The general lysosomal storage disease musculoskeletal lesion animal model constructed by the invention is used for researching the common pathogenesis of lysosomal storage disease musculoskeletal related lesions, and can also be used for screening, identifying and evaluating general medicines for the diseases.
The technical scheme of the invention is as follows:
a method for constructing a general lysosomal storage disease musculoskeletal lesion animal model, comprising the steps of:
(1) ConstructionChmp5 fl/fl Mice:
(S1) willChmp5As knockout sequences, the sequences of exons 4 and 5 and homologous sequences at both ends are cloned into a target vector;
(S2) injecting Cas9, gRNA and target vector together into fertilized eggs of mice, transplanting the fertilized eggs after injection into surrogate mice;
(S3) carrying out genotype identification on the born mice, and screening the mice successfully edited by genes, namely constructing to obtainChmp5 fl/+ Mice and methods of using the sameChmp5 fl/fl A mouse;
(2) Constructing an animal model:
by using the saidChmp5 fl/fl The mice are hybridized with the Cre mice with the specificity of osteoblast lineage cells, and the universal lysosomal storage disease musculoskeletal lesion animal model is constructed and obtained.
In step (S1), theChmp5The sequence of the exon 4 is shown as SEQ ID NO. 1;
the saidChmp5The sequence of exon 5 of (2) is shown in SEQ ID NO. 2.
The homologous sequences at the two ends are as follows: the 5 'end sequence is shown as SEQ ID NO.3, and the 3' end sequence is shown as SEQ ID NO. 4;
the target carrier is as follows: mammalian expression vector pSK+ comprising a loxp site.
In step (S2), cas9, gRNA and target vector are injected together into mouse fertilized eggs for CRISPR/Cas9 mediated gene editing.
In step (S3), the obtainedChmp5 fl/+ After the mice are mated with each other, obtainChmp5 fl/fl And (3) a mouse.
In the step (S3), the 5'-loxP site is 1807bp, the 3' -loxP site is 5836bp, and the conditional knockdown sequence is 1800bp.
In step (2), the osteoblast lineage cell specific Cre mouse isCtsk Cre Mice orDmp1 Cre And (3) a mouse.
When the osteoblast lineage cell specific Cre mice areCtsk Cre The specific construction method for the mice is as follows:
(A1) By using the saidChmp5 fl/fl Mice and methods of using the sameCtsk Cre The mice were mated with each other and,obtainingChmp5 fl/+ ;Ctsk Cre Heterozygous knockout mice;
(B1) Using the obtainedChmp5 fl/+ ;Ctsk Cre Heterozygous knockout miceChmp5 fl/fl Mating the mice to obtainChmp5 fl /fl ;Ctsk Cre The homozygote mice are the general lysosomal storage disease musculoskeletal lesion animal model.
When the osteoblast lineage cell specific Cre mice areDmp1 Cre The specific construction method for the mice is as follows:
(A2) By using the saidChmp5 fl/fl Mice and methods of using the sameDmp1 Cre Mating the mice to obtainChmp5 fl/+ ;Dmp1 Cre Heterozygous knockout mice;
(B2) Using the obtainedChmp5 fl/+ ;Dmp1 Cre Heterozygous knockout miceChmp5 fl/fl Mating the mice to obtainChmp5 fl /fl ;Dmp1 Cre The homozygote mice are the general lysosomal storage disease musculoskeletal lesion animal model.
The method for identifying the genotype of the mouse by PCR comprises the following steps:
(A3) 100-200 microliter of tissue DNA lysate is added to each mouse tail (2-5 mm);
(B3) Incubation at 56 ℃ for 2 hours or overnight;
(C3) Incubating at 98-100deg.C for 5 min, inactivating proteinase K;
(D3) Centrifugation at 12000rpm for 20 minutes, transferring the supernatant to a new centrifuge tube, and 2. Mu.l of supernatant was used for each PCR reaction;
（E3）Chmp5the PCR primer sequences were: forward 5'-CCCTTCTTGTTTCTGCTTT TAGAGA-3', reverse 5'-TCCAGGAAATCGGCAAATGATTCT-3'; the reaction conditions were as follows:
| step (a) | Temperature (temperature) | Time | Cycle number |
| Preliminary denaturation | 94 ℃ | 3. Minute (min) | |
| Denaturation (denaturation) | 94 ℃ | 30. Second of | |
| Annealing | 62 ℃ | 35. Second of | 35 x |
| Extension | 72 ℃ | 35. Second of | |
| Final extension | 72 ℃ | 5. Minute (min) |
(F3) The PCR product was run on a 1% agarose gel, the 212bp band was the mutant gene and the 151bp band was the wild-type gene.
The method constructs the obtained general lysosomal storage disease musculoskeletal lesion animal model.
The present inventors have found in long-term studies that charged polycystic protein 5 (charged multivesicular body protein, CHMP 5) is one of the members of protein complex III (ESCRT-III) required for endocytosis sorting, and plays a key role in transporting cell surface receptor ligand protein complexes, biomacromolecules, etc. to lysosomes for degradation. The ESCRT-III protein complex plays a key role in transportation and maturation after synthesis of lysosomal hydrolase, so that abnormal functions of the ESCRT-III protein complex can directly influence the activity and normal functions of lysosomal enzyme.
A mouseChmp5The gene is located on chromosome 4, has a full length of 16.9kb, contains 8 exons and encodes a protein consisting of 219 amino acids. The inventors of the present application found through studies that conditional knockouts in mouse osteoblast-associated cellsChmp5Gene [ (B/C)Chmp5 fl/fl ;Ctsk Cre AndChmp5 fl/fl ;DMP1 Cre ) Lysosomal function can be severely affected, causing intracellular lysosomal storage leading to early onset musculoskeletal and joint lesions, which can mimic musculoskeletal lesions of human lysosomal storage disorders.
Because the ESCRT-III protein complex in which CHMP5 participates plays a role in the transport and maturation of all lysosomal hydrolases, knocking out CHMP5 affects the normal function of all lysosomal enzymes, and the genetically engineered mice of this strain can be used as a universal animal model for studying the common pathogenesis of lysosomal storage disease musculoskeletal lesions and for screening universal therapeutic drugs.
Therefore, the invention provides a brand new method for conditional knocking out CHMP5 genes to construct an animal model of universal lysosomal storage disease based on the researchChmp5 germline knockout mice die from early embryonic development and cannot be born normally). Specifically, conditional knockdown of CHMP5 in osteoblast-related cells can construct an animal model of universal lysosomal storage disease musculoskeletal lesions.
First, conditional knockdownChmp5The gene is realized by a Cre-LoxP recombinase system. Cre is a site-specific recombinase which mediates specific recombination between LoxP sites, so that the gene sequence is deleted or recombined. The LoxP sequence consists of two repeated sequences of 13bp in opposite directions and a middle 8bp interval sequence, and the 8bp interval sequence determines the direction of LoxP. When two LoxP sites on the DNA chain are consistent in direction, cre recombinase can cut off sequences between the two LoxP sites, so that a truncated sequence of a frame-shifting (out-of-frame) can be generated on the gene, and gene knockout is realized.
Conditional knockdown in osteoblast-associated cellsChmp5The gene is Cre mouse specific to osteoblast lineage cellsChmpGenetically engineered mice carrying LoxP on both sides of gene exons 4 and 5Chmp5 fl/fl ) Hybridization, preparationChmp5 fl/fl ; Ctsk Cre AndChmp5 fl/fl ;Dmp1 Cre mice, thereby achieving conditional knockdown of CHMP5 gene in osteoblast-associated cells.
The inventors of the present application have found for the first time that conditional knockdown of CHMP5 in osteoblast-related cells can cause severe musculoskeletal lesions, which are caused by lysosomal storage and dysfunction in the cells, and thus can be used as animal models of lysosomal storage disease musculoskeletal lesions.
The inventors have also found that, not limited to musculoskeletal tissue, knocking out CHMP5 in other tissues like the nervous system should also be able to cause the associated pathological changes of the enzyme-body storage disorder. Meanwhile, the CHMP5 gene is not limited to animal models, and the CHMP5 gene can be knocked out in cells to construct a general in vitro cell model or an organoid model of lysosomal storage diseases.
The beneficial effects of the invention are as follows:
(1) The invention provides a general lysosomal storage disease musculoskeletal lesion animal modelPrevious constructionChmp5 fl/fl Mice, reuse the saidChmp5 fl/fl The mouse is hybridized with the Cre mouse with the specificity of the osteoblast lineage cells, and the universal lysosomal storage disease musculoskeletal lesion animal model is constructed and obtained, so that the CHMP5 gene is conditionally knocked out in the osteoblast related cells, and the method can be used for researching the common pathogenesis of the musculoskeletal related lesions of the lysosomal storage disease and can also be used for screening, identifying and evaluating universal medicines for the diseases.
(2) The method of the invention is constructedChmp5 fl/fl ;Ctsk Cre AndChmp5 fl/fl ;Dmp1 Cre the animal model of the musculoskeletal lesions of the lysosomal storage diseases is stable, the incidence rate is 100 percent, the musculoskeletal symptoms of mice are early, the lesions are typical and comprise extensive periosteum and endosteal hyperplasia, progressive joint mobility reduction and short stature, and the pathological manifestations can more comprehensively simulate musculoskeletal related lesions of the human lysosomal storage diseases.
(3) The animal model constructed by the method plays an important role in the transportation and maturation process of lysosomal hydrolase due to the ESCRT-III protein complex participated by CHMP5, so that the animal model disclosed by the invention can simulate musculoskeletal expression of almost all lysosomal storage diseases, is the only universal animal model capable of being used for researching the common pathogenesis of musculoskeletal lesions of the lysosomal storage diseases and evaluating drug screening at present, and increases an important tool for clinical etiology research, drug screening, verification and the like.
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In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the mediation of the Cre-LoxP system described in example 1Chmp5Simple antigen for gene knockoutB, managing a diagram;
FIGS. 2A-C show as describedChmp5 fl/fl ;Ctsk Cre Homozygote mouse animal model and littermatesChmp5 fl/fl Control plots of mice; wherein A isChmp5 fl/fl ;Ctsk Cre Gene knockout mouseChmp5 fl/fl The general image of the control mice shows that the gene knockout mice have smaller body types, sparse hair and strong proliferation of tail joints; b isChmp5 fl/fl ;Ctsk Cre Gene knockout mouseChmp5 fl/fl The x-ray image of the hind limb of the control mouse shows that the bone surface of the gene knockout mouse is widely subjected to hyperosteogeny at the near joint; c isChmp5 fl/fl ;Ctsk Cre Gene knockout mouseChmp5 fl/fl And (5) comparing the micro-CT images of the ankle joints of the mice to display the wide hyperosteogeny around the bone joints of the gene knockout mice.
FIGS. 3A-E show the process described in example 2Chmp5 fl/fl ;Dmp1 Cre Schematic representation of musculoskeletal lesions in homozygous mice. A is different agesChmp5 fl/fl ;Dmp1 Cre Gene knockout miceChmp5 fl/fl The x-ray image of hind limbs of the control mice shows that the bones of the gene knockout mice gradually expand and expand along with time (shown by arrows); b is 10 weeks oldChmp5 fl/fl ;Dmp1 Cre Gene knockout miceChmp5 fl/fl Comparing the general image and the maximum inner diameter and the maximum outer diameter of the femur of the control rat; c is 10 weeks oldChmp5 fl/fl ;Dmp1 Cre Gene knockout miceChmp5 fl/fl Quantifying the thickness of the femur cross section micro-CT image and the cortical bone of the control mouse; d is 10 weeks oldChmp5 fl/fl ;Dmp1 Cre Gene knockout miceChmp5 fl/fl Controlling the femoral histological image of the mouse to display the inner cortical hyperplasia of the mouse with the gene knocked out; e is 10 weeks oldChmp5 fl/fl ;Dmp1 Cre Gene knockout miceChmp5 fl/fl Hind limb abduction general images and muscle weight and function measurements of control mice.
FIGS. 4A and B show lysosomal storage in CHMP5 gene knockout osteoblast progenitor cells isolated from animal models. A is shown using lysosomal tracerChmp5The fluorescence intensity of the tracer in the gene knocked-out osteoblast progenitor cells is increased compared with that in normal cells, indicating lysosomal storage; b is a cell immunostaining lysosomal protein LAMP1, showing thatChmp5The number and size of lysosomes in the knocked-out osteoblast progenitor cells were both increased over normal cells, indicating lysosomal storage.
FIGS. 5A and B show lysosomal dysfunction in CHMP5 gene knockout osteoblastic progenitor cells isolated from animal models. A is a transmission electron microscope image, and showsChmp5The osteoblast progenitor cells with the gene knocked out have more lysosome-like structures with high electron density and low electron density, and the high electron density images indicate degradation dysfunction of the lysosomes; b is a fluorescent tracing technique, displayChmp5The function of the osteoblast progenitor cells with the knocked-out genes for degrading the EGF ligand receptor protein complex on the cell surface is reduced compared with that of normal cells.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
The following describes the above technical scheme in detail with reference to specific embodiments.
The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 constructionChmp5 fl/fl A mouse
The present embodiment provides a constructionChmp5 fl/fl The construction method of the mice comprises the following specific steps:
(S1) willChmp5As knockout sequences, the sequences of exons 4 and 5 and homologous sequences at both ends are cloned into a target vector;
wherein the saidChmp5The sequence of exon 4 of (2) is shown as SEQ ID NO.1, and specifically:
GTATGAGCAACAGCGAGACAACCTGGCCCAACAGTCCTTTAACATGGAGCAAGCTAATTACACCATCCAGTCACTAAAGGACACCAAGACCACG；
the saidChmp5The sequence of exon 5 of (2) is shown as SEQ ID NO.2, specifically:
GTTGATGCCATGAAGTTGGGAGTAAAGGAAATGAAGAAGGCATATAAGGAAGTAAAAATTGACCAGATTGAG；
the homologous sequences at the two ends are as follows: the 5' end sequence is shown as SEQ ID NO.3, and concretely comprises:
GTAGAGGTTCCCAGATTCTGAAAGCGGGAGGCTGGTGGGCTGTTGTAAATGGCTTCTAACATGAGGACATGATAGCGGATGAACGTGTTGTATTTAAATGGAAAGCAACTGGATATACACAGTGCGCTCTGTTGGGGCTGGAAGTGGAAGTACAATGCCTAGGGAGAGGAAGAGGAATTGAAGATACAGAGCATGGGTTGCAAAGGGCGCTAAGATGTACCAGCTGGGCATTAGAATTATCTGTGCTCGAAGCTTGAAGAGGAAGAGTAGAGTCAAGGAGCCTCCACATCTGCCATCTTGCAGAAGAAACATGTAGGTGCAGGAGTGTGAGCATAGGGGAGACTTCTTGTGCCATCTGCGTTCCCTTCTTTTAATTAAGTCTGTTTTTCTCCTCAGAACATGGTCAAACAGAAAGCCCTGAGAGTTTTAAAGCAAAAGCGGATGTAAGTCATATGCTTCATCTCCCTCCTAAGAAATCAGAAAGTGAAGAGGCCTTCCATTCCTTCCGGCTTTCATTTCTTCTCAGTAGTCTTGATTTCATTACAAATGTGCAAAAGCTTCCAATGTTCCGAGTTACTACCTAAGCTTCAGCAGCCACGTGGGATCACTTTGGCTGGCAAGGTCGAGATGGTTTGGGTCTCCTGTGGTTGAGTTAGGATGAGTGTTTCGGTTTTGTTTGTAAAGTTAGGAGTGCTAAATATTCATTCTCTCTCACCAGAATAAATGTGAAGGTTCACAATTTATTTTCAATTACTATGTCCCAGTGTTTGTGCGAAACCCCAGTTTATCTGTGGTGCTCACCCATGTGGTCTGCTCAGCCTATGAGATCATCCATGGAGTTGTAGACTCGAGGCATAATCAGCTTGTAGGAAACCAGGAAAAGTGTGATGGCTTTGTTTCAATTTTAAAATGACTTTGTAACAGTCAGATGCTGTCTTCCATTTTTAAAAAATACATTAAAATCACATTGGAAAAAAAAACCTAATAATACCTAGAATGGCCAGCATGTATTACAACTGTTGCCTTCTTGGTGGCAATTGGTTTTAAAGTATTTTGGTTTTCTTGTGGAAGGAATTTGGCTTTGATGGCCTGGACCTGTTATCCTAGCATTTGGGAGGTAGAGGACCCGGAACCTATGAGCATTGACTACATAGAGAAATCAAGACTAGCCTTGGCTACATAAGACCCTGTCTCAAATAAATAAATAAACCAAGCAGGGTGGCACACAACCTGTAGACCTAGCACTTGGTATATGGAGGCAAGAACATCAGGGGTTCAAAGCTACCCTGGGCTAGTAAATTCTAGAGTAACTTGGGCTACATGAATCCCTGCCTCAAAAATAAATAAATAAATGGAATGTAGATTTATTGCAGAAACTTGGAACATATATCAAACAGAGGTAAAGAAAATTTATCTAGATTCTTAGGAAACTGAAATTATGTGTCTTTTATATAAGCTGTCGTCTTGCCCCTTCTTGTTTCTGCTTTTAGAGACAAGGTTCTCTGTGTACTCCACGCTGGCCTCAAGCTCTAGTCCCCTGGCTCAGTCCCCCAGTTGCTAGGATTATAACCTTGTGCCACGA；
the 3' end sequence is shown as SEQ ID NO.4, and concretely comprises:
AAGAGCAGCACTGCTCTTAACCACTACACCATCTTCCCACTCAAGCTTCACTTTGCTTCTGAGGCAGGATGTCATTCTGTAGCTTAGGCTCTTGTAGAATGTATTATATCTCCTAGGCTAGCCCTGCACTTGTGTCAGTCCTGCCTCAGCCCCTGAAGTGCTAGCCTTGCCTTCAACCTCATTCTGGCTTTTGGCTTTTTGGATGCCTCCCTATCTCCTTTCCATTTTCCTTGTCTGCCCCTAATAGATTGCCAGCACGACACACTTCATAAATGTATCCTTTGCCTGTGCTATACACTTGGAACTCTTAGGCTTATTTTTCTAATATAAATTAAAATATTTTTATTGTTTTATAGTTCTAATTTTTGAATTTTTATTTGCATGTTTAGCTGTTTTTATTCTTTTTATTAAAAAAAATTGAAAAAAAAAAAAGAGAGAGAGAAGACCGGACATGTTGTCGCACGCCTTTGATCCTAGCACTTGGGAGGCAGAGGCAGGCAGATTTCTGAGTTCGAGGCCAGCCTGGTTTACAGAGTGAGTTCCAGGACAGCCAGGGATACACAGAGAAACCCTGTCTCGAAAAACCAAAAAAAACCAACCAAACAAACAAAAAGACTTTGGGCCAGGTTAATGGGGTACACCTTTTGTCCCAGCACTCCAGAGGCAGAAGCAGGAGGATCTCCGTGAGTTTGACATCAGCCTGGTCTACAGAGTGAGTTCTCGAACAGCTAGGGCTACACAGAGAGACCCTTTCTCAAAAAAGATCATTTTTATTTTTCAGTATGTGTATATCTGTGTGTTTGCTTTTTGTGTTTATGGAGGCCAGAAGAGAACAGATCCCAGCAGCTGGAGAGACAGGCAGTTGTGAGCTGACTGTTGCGGCTGCTGGAAGCCACACTCAGATCTTCAGCAAGAGCAGTCCACAATTTTAGCCAATGAGACACCTACCCATCCCAACTCCCTATTTACATTTTTCTATCTTTTTATTTCACAGAACATTAGGGCATCTATGCTTTGTAGATTAGCACTTCTGTTTGTGTTGCTTTTGTTTATCTAGTATTGATCTTAAAGGTATATTAACCTGATACTTTGCATGGTAACTTTCTTTTCTCTTGCTGTGTCTGTACTAGACTATGTAACTAGAAACTGTGTTCATAGGCTGGGTTATGGTTGCAGGCCTATAATCTCAAAAGTCAGAGGCAGAAGAATTATAAGTTCAAGGCTTGGGGCTGGAGAGATGGCCCAGAGGTTAAGAGCACTGACTGCTCTTCCAGAGGTCCTGAGTTCAGTTCCCAGCAACCACATGGTGGCTCACAACCATCTGTAATGGAATCCGATGCCCTCTTCTGTGTGTCTGAAGACAGCTACAGTATGCTCATATAAAATAAATAAGTTCAGGGCCGGC；
the target carrier is as follows: a mammalian expression vector pSK+ comprising a loxp site;
(S2) injecting Cas9, gRNA and target vector together into fertilized eggs of mice, transplanting the fertilized eggs after injection into surrogate mice; performing CRISPR/Cas9 mediated gene editing;
(S3) carrying out genotype identification on the born mice, and screening the mice successfully edited by genes, namely constructing to obtainChmp5 fl/+ Mice and methods of using the sameChmp5 fl/fl A mouse;
sequencing analysis is adopted to identify that the 5'-loxP insertion site is 1807bp, the 3' -loxP insertion site is 5836bp, and the conditional knocked-out sequence is 1800bp;
the obtained productChmp5 fl/+ After the mice are mated with each other, obtainChmp5 fl/fl And (3) a mouse.
Example 2 constructionChmp5 fl/fl ;Ctsk Cre Homozygous mice
The embodiment is based on the followingChmp5 fl/fl The method comprises the steps of constructing a universal lysosomal storage disease musculoskeletal lesion animal model by a mouse, wherein the universal lysosomal storage disease musculoskeletal lesion animal model is as followsChmp5 fl/fl ;Ctsk Cre Homozygous mice, realization ofChmp5The principle of gene knockout is shown in figure 1, and specifically comprises the following steps:
(A1) By using the saidChmp5 fl/fl Mice and methods of using the sameCtsk Cre Mating the mice to obtainChmp5 fl/+ ;Ctsk Cre Heterozygous knockout mice;
(B1) Using the obtainedChmp5 fl/+ ;Ctsk Cre Heterozygous knockout miceChmp5 fl/fl Mating the mice to obtainChmp5 fl /fl ;Ctsk Cre The homozygote mice are the general lysosomal storage disease musculoskeletal lesion animal model.
The method for identifying the genotype of the mouse by PCR comprises the following steps:
(A3) 100-200 microliter of tissue DNA lysate is added to each mouse tail (2-5 mm);
(B3) Incubation at 56 ℃ for 2 hours or overnight;
(C3) Incubating at 98-100deg.C for 5 min, inactivating proteinase K;
(D3) Centrifugation at 12000rpm for 20 minutes, transferring the supernatant to a new centrifuge tube, and 2. Mu.l of supernatant was used for each PCR reaction;
（E3）Chmp5the PCR primer sequences were: forward 5'-CCCTTCTTGTTTCTGCTTT TAGAGA-3', reverse 5'-TCCAGGAAATCGGCAAATGATTCT-3'; the reaction conditions were as follows:
| step (a) | Temperature (temperature) | Time | Cycle number |
| Preliminary denaturation | 94 ℃ | 3. Minute (min) | |
| Denaturation (denaturation) | 94 ℃ | 30. Second of | |
| Annealing | 62 ℃ | 35. Second of | 35 x |
| Extension | 72 ℃ | 35. Second of | |
| Final extension | 72 ℃ | 5. Minute (min) |
(F3) The PCR product was run on a 1% agarose gel, the 212bp band was the mutant gene and the 151bp band was the wild-type gene.
The saidChmp5 fl/fl ;Ctsk Cre Homozygote mouse animal model and littermatesChmp5 fl/fl Mice were subjected to controls (as shown in FIGS. 2A-C), FIG. 2A beingChmp5 fl/fl ;Ctsk Cre Gene knockout mouseChmp5 fl/fl A general image of the control mice, from which it can be seen:Chmp5 fl/fl ;Ctsk Cre homozygous knockout mice are more littermateChmp5 fl/fl The body type of the control mice is obviously reduced, the hair is sparse, and the joint hyperplasia of the tail is strong. FIG. 2B is a schematic view ofChmp5 fl/fl ;Ctsk Cre Gene knockout mouseChmp5 fl/fl The x-ray image of the hind limb of the control mouse shows that the bone surface of the gene knockout mouse is widely subjected to hyperosteogeny at the near joint; FIG. 2C isChmp5 fl /fl ;Ctsk Cre Gene knockout mouseChmp5 fl/fl The micro-CT image of the ankle joint of the control mouse shows that the bone joint of the gene knockout mouse has wide hyperosteogeny, as can be seen from the figure 2B and the figure 2C,Chmp5 fl/fl ;Ctsk Cre homozygous knockoutThe mice gradually develop periosteum hyperostosis after birth, and the mice are gradually aggravated along with growth, and obvious joint rigidity and limited movement begin to appear about 6 weeks, so that the growth and development of the mice are seriously affected.
Example 3 constructionChmp5 fl/fl ;Dmp1 Cre Homozygous mice
The embodiment is based on the followingChmp5 fl/fl The method comprises the steps of constructing a universal lysosomal storage disease musculoskeletal lesion animal model by a mouse, wherein the universal lysosomal storage disease musculoskeletal lesion animal model is as followsChmp5 fl/fl ;Dmp1 Cre Homozygous mice, realization ofChmp5The principle of gene knockout is as shown in figure 1, and the construction method specifically comprises the following steps:
(A2) By using the saidChmp5 fl/fl Mice and methods of using the sameDmp1 Cre Mating the mice to obtainChmp5 fl/+ ;Dmp1 Cre Heterozygous knockout mice;
(B2) Using the obtainedChmp5 fl/+ ;Dmp1 Cre Heterozygous knockout miceChmp5 fl/fl Mating the mice to obtainChmp5 fl /fl ;Dmp1 Cre Homozygous mice were genotyped by PCR, and the specific identification method was confirmed as in example 2Chmp5 fl/fl ;Dmp1 Cre The homozygote mice are the general lysosomal storage disease musculoskeletal lesion animal model.
DMP1 is expressed in endosteal progenitor cells, osteoblasts and osteocytes, as shown in FIG. 3Chmp5 fl/fl ;Dmp1 Cre Musculoskeletal lesions of homozygous mice are schematically represented in FIGS. 3A-E, and FIG. 3A shows musculoskeletal lesions of different agesChmp5 fl/fl ;Dmp1 Cre Gene knockout miceChmp5 fl/fl The x-ray image of hind limbs of control mice can be seen from the figure:Chmp5 fl/fl ; Dmp1 Cre homozygous knockout mice were from 3 weeksBone gradually proliferates and swells at age 1 and bone lesions are very severe. Further analysis of bones of 10-week-old mice was performed as shown in FIG. 3B as 10-week-old miceChmp5 fl/fl ;Dmp1 Cre Gene knockout miceChmp5 fl/fl Comparing the general image and the maximum inner diameter and the maximum outer diameter of the femur of the control rat; as can be seen from the figures of the drawing,Chmp5 fl/fl ;Dmp1 Cre bone of homozygous knockout mice swelled significantly; as shown in FIG. 3C as 10 weeks of ageChmp5 fl/fl ;Dmp1 Cre Gene knockout miceChmp5 fl/fl Control rat femur cross section micro-CT image and cortical bone thickness quantification, FIG. 3D shows 10 weeks of ageChmp5 fl /fl ;Dmp1 Cre Gene knockout miceChmp5 fl/fl Control rat femur histological image, histological analysis displayChmp5 fl/fl ; Dmp1 Cre Hyperosteogeny in homozygous knockout mice occurs mainly in endosteal membranes. In addition, DMP1 is also expressed in skeletal muscle cells,Chmp5 fl/fl ;Dmp1 Cre the skeletal muscle volume, weight and function of homozygous knockout mice also gradually decrease, resulting in a great limitation of limb movement, as shown in fig. 3E, which is 10 weeks oldChmp5 fl/fl ;Dmp1 Cre Gene knockout miceChmp5 fl/fl The hind limb abduction general image and the measurement result of muscle weight and function of the control mice can be seen: these musculoskeletal manifestations better mimic musculoskeletal lesions of human lysosomal storage diseases.
As shown in fig. 4, by isolating CHMP5 knockdown osteoblast progenitor cells in animal models, using lysosomal tracer detection, the fluorescence intensity of lysosomal tracer in CHMP5 knockdown osteoblast progenitor cells was found to be significantly increased, indicating lysosomal storage (fig. 4A); further detection of the cell lysosomal protein LAMP1 by cellular immunofluorescence revealed LAMP1 + The number and size of lysosomal vesicles increased significantly in CHMP5 knockdown osteoblast progenitor cells compared to normal cells (fig. 4B) Knocking out the CHMP5 gene has also been shown to result in lysosomal storage in osteoblastic progenitor cells.
Meanwhile, as shown in fig. 5, transmission electron microscopy further observed a large amount of lysosomal storage in CHMP5 knockdown cells, partially containing also high electron density of non-decomposed biomacromolecules, indicating that knocking down CHMP5 gene resulted in reduced degradation function of osteoblast progenitor lysosomes (fig. 5A). Further, using fluorescent tracer technology, a significant impairment of the function of lysosomal degradation of cell surface receptor ligand protein complexes within CHMP5 knockdown osteoblast progenitor cells was observed (fig. 5B).
The above changes in the number, volume and function of CHMP5 knockouts of intracellular lysosomes closely resemble those in human lysosomal storage disorders. Importantly, more lysosomal enzymes are involved in CHMP5 knocked-out cells, so that the method is an animal model of severe lysosomal storage disease musculoskeletal lesions, and has wider application range and stronger practicability.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. A method for constructing a universal lysosomal storage disease musculoskeletal lesion animal model, comprising the steps of:
(1) ConstructionChmp5 fl/fl Mice:
(S1) willChmp5As knockout sequences, the sequences of exons 4 and 5 and homologous sequences at both ends are cloned into a target vector;
(S2) injecting Cas9, gRNA and target vector together into fertilized eggs of mice, transplanting the fertilized eggs after injection into surrogate mice;
(S3) genotyping the born mice, screening mice successfully edited with genes, i.eConstructed to obtainChmp5 fl/+ Mice and methods of using the sameChmp5 fl/fl A mouse;
(2) Constructing an animal model:
by using the saidChmp5 fl/fl The mice are hybridized with the Cre mice with the specificity of osteoblast lineage cells, and the universal lysosomal storage disease musculoskeletal lesion animal model is constructed and obtained.
2. The method for constructing a universal lysosomal storage disease musculoskeletal disease animal model according to claim 1, wherein in step (S1), theChmp5The sequence of the exon 4 is shown as SEQ ID NO. 1;
the saidChmp5The sequence of exon 5 of (2) is shown in SEQ ID NO. 2.
3. The method for constructing a universal lysosomal storage disease musculoskeletal disease animal model according to claim 1, wherein the homologous sequences at the two ends are: the 5 'end sequence is shown as SEQ ID NO.3, and the 3' end sequence is shown as SEQ ID NO. 4;
the target carrier is as follows: mammalian expression vector pSK+ comprising a loxp site.
4. The method of claim 1, wherein in step (S2), cas9, gRNA and target vector are injected together into a mouse fertilized egg for CRISPR/Cas9 mediated gene editing.
5. The method for constructing a universal lysosomal storage disease musculoskeletal disease animal model according to claim 1, wherein in step (S3), the obtainedChmp5 fl/+ After the mice are mated with each other, obtainChmp5 fl/fl And (3) a mouse.
6. The method for constructing a musculoskeletal lesion animal model for general lysosomal storage diseases according to claim 1, wherein in the step (S3), the 5'-loxP insertion site is 1807bp, the 3' -loxP insertion site is 5836bp, and the conditional knockdown sequence is 1800bp, which is identified by sequencing analysis.
7. The method of claim 1, wherein in step (2), the osteoblast lineage cell specific Cre mice areCtsk Cre Mice orDmp1 Cre And (3) a mouse.
8. The method of claim 7, wherein when the osteogenic lineage cell specific Cre mouse isCtsk Cre The specific construction method for the mice is as follows:
(A1) By using the saidChmp5 fl/fl Mice and methods of using the sameCtsk Cre Mating the mice to obtainChmp5 fl/+ ;Ctsk Cre Heterozygous knockout mice;
(B1) Using the obtainedChmp5 fl/+ ;Ctsk Cre Heterozygous knockout miceChmp5 fl/fl Mating the mice to obtainChmp5 fl/fl ;Ctsk Cre The homozygote mice are the general lysosomal storage disease musculoskeletal lesion animal model.
9. The method of claim 7, wherein when the osteogenic lineage cell specific Cre mouse isDmp1 Cre The specific construction method for the mice is as follows:
(A2) By using the saidChmp5 fl/fl Mice and methods of using the sameDmp1 Cre Mating the mice to obtainChmp5 fl/+ ;Dmp1 Cre Heterozygous knockout mice;
(B2) Using the obtainedChmp5 fl/+ ;Dmp1 Cre Heterozygous knockout miceChmp5 fl/fl Mating the mice to obtainChmp5 fl/fl ;Dmp1 Cre The homozygote mice are the general lysosomal storage disease musculoskeletal lesion animal model.
10. A universal lysosomal storage disorder musculoskeletal lesion animal model constructed according to the method of any one of claims 1-9.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160237455A1 (en) * | 2013-09-27 | 2016-08-18 | Editas Medicine, Inc. | Crispr-related methods and compositions |
| CN107114311A (en) * | 2017-04-26 | 2017-09-01 | 西北工业大学 | The structure for the mouse model that MACF1 genes are knocked out in Gegenbaur's cell conditional and application |
| CN109694881A (en) * | 2018-12-19 | 2019-04-30 | 首都医科大学附属北京口腔医院 | The construction method of Ano5 gene knock-out mice model |
| US20210299169A1 (en) * | 2020-02-18 | 2021-09-30 | Unm Rainforest Innovations | Galectin-3 Governs and Coordinates ESCRT and Autophagic Responses During Endomembrane Damage |
| WO2023004847A1 (en) * | 2021-07-26 | 2023-02-02 | 浙江树人学院（浙江树人大学） | Mouse premature senility model, construction method therefor, and application thereof |
-
2024
- 2024-01-18 CN CN202410069550.2A patent/CN117587069A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160237455A1 (en) * | 2013-09-27 | 2016-08-18 | Editas Medicine, Inc. | Crispr-related methods and compositions |
| CN107114311A (en) * | 2017-04-26 | 2017-09-01 | 西北工业大学 | The structure for the mouse model that MACF1 genes are knocked out in Gegenbaur's cell conditional and application |
| CN109694881A (en) * | 2018-12-19 | 2019-04-30 | 首都医科大学附属北京口腔医院 | The construction method of Ano5 gene knock-out mice model |
| US20210299169A1 (en) * | 2020-02-18 | 2021-09-30 | Unm Rainforest Innovations | Galectin-3 Governs and Coordinates ESCRT and Autophagic Responses During Endomembrane Damage |
| WO2023004847A1 (en) * | 2021-07-26 | 2023-02-02 | 浙江树人学院（浙江树人大学） | Mouse premature senility model, construction method therefor, and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| MATTHEW B. GREENBLATT: "CHMP5 controls bone turnover rates by dampening NF-B activity in osteoclasts", 《J. EXP. MED.》, vol. 201, no. 8, 20 June 2015 (2015-06-20), pages 1283 - 1301 * |
| XIANPENG GE: "The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network", 《BIORVIX》, 4 August 2020 (2020-08-04), pages 1 - 63 * |
| 汪冰: "《生物化学》", 30 November 2020, 东北大学出版社, pages: 365 - 367 * |
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