KR20170001021A - Sarconia model mice, preparation method thereof and screening method of sarconia using them - Google Patents
Sarconia model mice, preparation method thereof and screening method of sarconia using them Download PDFInfo
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Abstract
Description
The present invention relates to an animal model for myopenia, a method for producing the animal model, and a screening method for treating an agent for treating myopenia.
Sarcopenia is a typical degenerative muscle and neurological disorder in which muscle mass and muscle strength decrease together with age. The clinical manifestations of myopenia are more prevalent in males than females, and begin to appear from 50 years of age or older. In 80 years or older, one in two has myopenia, and muscle mass, mobility and muscle strength are significantly reduced, I have symptoms. In addition to the myopenia observed in the elderly, myopenia secondary to various diseases is known to be an important factor that makes the improvement of the disease difficult.
In other words, myopenia is a group of diseases expressed when both skeletal muscle function and skeletal muscle function are satisfied. Primary sarcopenia due to aging and secondary sarcopenia due to various causes are present. Secondary causes include nutritional deficiencies, decreased activity, organ failure, drugs, inflammation or malignant diseases, and endocrine metabolic diseases.
In modern Korean society, the elderly population is rapidly increasing due to an increase in the average life expectancy of both men and women, and the proportion of the elderly living independently is increasing due to the nuclear family and the declining fertility rate. In the future, it is expected that serious medical and social costs will be induced by the explosive increase in the elderly population and the decrease of independence due to the aging-related myopenia. However, due to the complicated pathogenesis of myopenia, there is no common target of treatment and there is no proper drug, so active nutritional supplementation and exercise therapy is the only treatment. Therefore, in order to find a therapeutic target based on the common etiology, development of an animal model for myopenia and drug screening using this model is urgent.
Therefore, it is possible to predict the occurrence of myopenia according to the desired time and age, 2) to understand the occurrence of myopenia due to the combined action of various causes, and 3) There is an urgent need for an animal model that can be applied to the development of therapeutic agents for treating myopenia and the treatment of diseases.
CRIF1 (CR6-interacting factor 1) is a mitochondrial protein that binds to Gadd45 (growth arrest and DNA damage inducible). The deficiency of CRIF1 reduces the stability of the respiratory complex present in the mitochondrial inner membrane, resulting in reduced mitochondrial respiration rates, increased reactive oxygen species and structural changes in the mitochondrial inner membrane.
Cre-loxp is a system capable of conditionally destroying genes. When a Cre recombinase derived from bacteriophage P1 recognizes a loxp site, the target gene fragment flanked by the loxp sequence is removed, Destruction occurs. At this time, the Cre recombinase is activated by the tissue and cell-specific promoter located in front of the Cre transplant gene, and the target gene is inactivated in the designated tissue or cell. The Loxp sequence has 13 bases at the beginning and end are inversely related to each other, and the sequence at the center varies depending on the type of the loxp sequence.
The present inventors have studied to develop an animal model of myopenic animal by suppressing the mitochondrial respiratory chain in a muscle-specific manner in order to solve clinical untimely demand, and an animal model in which skeletal muscle cells specifically knock-out CRIF1 gene The amount of myocytes, the amount of exercise and the amount of mobility were decreased during growth compared to the control group.
It is an object of the present invention to provide an animal model for myopenia, a method for producing the animal model, and a method for screening for a therapeutic agent for myopenia.
In order to accomplish the above object, the present invention provides a sacropenia animal model in which CRIF1 (CR6 interacting factor 1) gene is knocked out specifically for muscle cells.
The present invention also provides a method for screening a CRIF1 loxp / loxp mouse in which a Loxp site is inserted next to the
Crossing said heterozygous knockout mouse and CRIF loxp / loxp mouse to obtain a second generation mouse; And
Selecting a CRIF1 homozygous knock out mouse having a genotype of CRIF1 loxp / loxp and MLC-cre M / + from the second generation mouse.
The present invention also relates to a method of treating a human hypersensitivity animal model of the present invention,
Measuring at least one of a muscle decline rate or an increase rate and a survival rate of the animal model after administration of the sample; And
And selecting a sample that improves at least one of a muscle reduction rate or an increase rate and a survival rate in comparison with a control group to which the sample is not administered.
The present invention also relates to a method of treating a human hypersensitivity animal model of the present invention,
Measuring the mobility of the animal model after administration of the sample; And
And screening a sample for improving mobility in comparison with a control group to which the sample is not administered.
The animal model of myopenic animal according to the present invention is characterized by a decrease in the amount of myocardial cells, the amount of exercise, and mobility of the myocardium at the time of birth without any abnormality in sex ratio and appearance, Model and screening for the prophylactic and therapeutic agent for myopenia.
FIG. 1A shows the structure of the vector used for animal model production and genotype of muscle cell-specific CRIF1 knockout mouse:
CRIF f / f : CRIF1 loxp / loxp mouse with Loxp site inserted next to
CRIF1 f / + MLC1f : CRIF loxp / +, MLC m / + heterozygous knock out mouse; And
CRIF1? Ssm: CRIF loxp / loxp, MLC m / + homozygous knock out mice.
FIG. 1B shows the increase in body weight due to the growth of muscle cell-specific CRIF1 knockout mice:
CRIF f / f : CRIF1 loxp / loxp mouse with Loxp site inserted next to
CRIF1 f / + MLC1f : CRIF loxp / +, MLC m / + heterozygous knock out mouse; And
CRIF1? Ssm: CRIF loxp / loxp, MLC m / + homozygous knock out mice.
FIG. 1C shows the survival rate of muscle cell-specific CRIF1 knockout mice according to their age. FIG.
CRIF f / f : CRIF1 loxp / loxp mouse with Loxp site inserted next to
CRIF1 f / + MLC1f : CRIF loxp / +, MLC m / + heterozygous knock out mouse; And
CRIF1? Ssm: CRIF loxp / loxp, MLC m / + homozygous knock out mice.
Figure 1d shows a phenotype at 13 weeks of control mice and muscle cell specific CRIF1 knockout mice:
CRIF f / f : CRIF1 loxp / loxp mouse with Loxp site inserted next to
CRIF1? Ssm: CRIF loxp / loxp, MLC m / + homozygous knock out mice.
FIG. 2a shows the result of confirming muscle mass in 13-week-old muscle cell-specific CRIF1 knockout mice:
CRIF f / f : CRIF1 loxp / loxp mouse with Loxp site inserted next to
CRIF1? Ssm: CRIF loxp / loxp, MLC m / + Tumor homozygous knock out mice;
Gas: Gastrocnemius; And
TA / EDL: Tibialis anterior / Extensor digitorum longus.
Figure 2b shows the weight of organs and muscles in 8-week-old muscle cell specific CRIF1 knockout mice:
CRIF f / f : CRIF1 loxp / loxp mouse with Loxp site inserted next to
CRIF1? Ssm: CRIF loxp / loxp, MLC m / + Tumor homozygous knock out mice;
iWAT: Inguinal white adipose tissue;
eWAT: epididymal white adipose tissue;
EDL: Extensor digitorum longus; And
Gas: Gastrocnemius.
Figure 2b is a graph depicting reduced phenotype and muscle bundle area of muscle cells in 8-week-old muscle cell specific CRIF1 knockout mice:
CRIF f / f : CRIF1 loxp / loxp mouse with Loxp site inserted next to
CRIF1? Ssm: CRIF loxp / loxp, MLC m / + homozygous knock out mice.
FIG. 3 shows the results of staining of succinate dehydrogenase in muscles of 8-week-old muscle cell-specific CRIF1 knockout mice:
CRIF f / f : CRIF1 loxp / loxp mouse with Loxp site inserted next to
CRIF1? Ssm: CRIF loxp / loxp, MLC m / + homozygous knock out mice.
FIG. 4 is a graph showing grip strength, mean latency, and mobility of 13-week-old muscle cell-specific CRIF1 knockout mice:
CRIF f / f : CRIF1 loxp / loxp mouse with Loxp site inserted next to
CRIF1? Ssm: CRIF loxp / loxp, MLC m / + homozygous knock out mice.
FIG. 5A is a graph showing electron microscopic examination of mitochondria in a long-toe extensor muscle of a muscle-cell-specific CRIF1 knockout mouse. FIG.
CRIF f / f : CRIF1 loxp / loxp mouse with Loxp site inserted next to
CRIF1? Ssm: CRIF loxp / loxp, MLC m / + homozygous knock out mice.
FIG. 5b shows the expression levels of representative proteins involved in mitochondrial respiratory chain complexes in muscle cell-specific CRIF1 knockout mice:
CRIF f / f : CRIF1 loxp / loxp mouse with Loxp site inserted next to
CRIF1? Ssm: CRIF loxp / loxp, MLC m / + Tumor homozygous knock out mice;
NDUFA9:
SDHA: succinate dehydrogenase complex, subunit A, flavoprotein variant;
UQCRC2: ubiquinol-cytochrome c
COX I: cyclooxygenase I; And
Tom40: translocase of the
Figure 4c shows an assembly abnormality of the mitochondrial respiratory chain complex in a muscle cell specific CRIF1 knockout mouse:
CRIF f / f : CRIF1 loxp / loxp mouse with Loxp site inserted next to
CRIF1? Ssm: CRIF loxp / loxp, MLC m / + Tumor homozygous knock out mice; And
Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ: Mitochondrial respiratory chain complex.
Hereinafter, the present invention will be described in detail.
The present invention provides a sacropenia animal model in which a CRIF1 (CR6-interacting factor 1) gene is knocked out specifically in a muscle cell.
The myopenia refers to a decrease in muscle mass and muscle strength due to aging, but is not limited thereto.
The myopenic animal model is preferably homozygote, but not limited thereto.
The present invention also provides a method for producing the animal model of myopenia.
The preparation method preferably includes but is not limited to the following steps:
1) CRIF1 loxp / loxp mice in which the Loxp site was inserted next to the
2) crossing the heterozygous knockout mouse and CRIF loxp / loxp mouse to obtain a second generation mouse; And
3) Selection of CRIF1 homozygous knock out mice having the genotype of CRIF1 loxp / loxp and MLC-cre M / + in the second generation mouse.
The CRIF1 gene is preferably represented by the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.
The CRIF1 homozygous knockout mice in step 3) have CRIF1 genetic defects in all pairs of homologous chromosomes, which have a genetic trait of CRIF1 - / - and no abnormality in sex ratio and appearance at birth, There are features that indicate symptoms.
In a specific embodiment of the present invention we crossed CRIF1 loxp / loxp mice and MLC-cre mice to generate a first generation CRIF1 heterozygous knock with a genotype of CRIF1 loxp / + and MLC-cre m / + out mice were obtained by crossing a heterozygous knockout mouse and a CRIF loxp / loxp mouse to obtain a second generation mouse, and a CRIF1 homozygous knockout knockout mouse having a genotype of CRIF1 loxp / loxp and MLC-cre M / + in the second generation mouse (homozygous knock out) mice were selected as myopenic animal models. The animal model of selected myopenidosis has lowered the amount of weight gain (see FIG. 1B), the amount of muscle cells, the amount of exercise, and the locomotor power (FIG. 2 FIG. 4). Since it exhibits intrinsic symptoms of myopenia, it can be usefully used as an animal model of myopenic progression and pathological mechanism studies.
The present invention also provides a method for screening for a prophylactic and therapeutic agent for myopenia using an animal model of myopenia comprising the following steps:
1) administering a sample to a myopenic animal model;
2) measuring at least one of a muscle decline rate or an increase rate and a survival rate of the animal model after administration of the sample; And
3) selecting a sample that improves at least one of the rate or rate of increase or survival of the muscle compared to the control without the sample.
The animal model of myopenidia in step 1) above refers to a CRIF1 homozygous knockout mouse in which CRIF1 gene deletion is specifically expressed in muscle cells.
The method of administering the sample in the step 1) may be appropriately selected from the conventional methods of administering the sample, such as oral administration, intravenous injection, subcutaneous administration, and intraperitoneal administration, and the dosage of the sample is appropriately determined depending on the administration method, You can choose.
The present invention also provides a method for screening for a prophylactic and therapeutic agent for myopenia using an animal model of myopenia comprising the following steps:
1) administering a sample to a myopenic animal model;
2) measuring the mobility of the animal model after administration of the sample; And
3) Selecting the samples that improve mobility compared to the control without the sample.
The animal model of myopenidia in step 1) above refers to a CRIF1 homozygous knockout mouse in which CRIF1 gene deletion is specifically expressed in muscle cells.
The sample of step 1) does not significantly affect the mobility of the control group, but is not limited thereto.
Since the myopenid animal model of the present invention shows that the amount of muscle cells, the amount of exercise, and the locomotor activity are decreased with the growth of the animal model, it can be usefully used for the screening of a prophylactic and therapeutic agent for myopenia.
Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.
However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.
Example 1 Preparation of muscle cell specific CRIF1 homozygous knockout mice
In order to prepare mice lacking the CRIF1 gene specifically for muscle cells, the following experiment was conducted. All mice were subjected to preclinical experimental center (LMO registration number: LML-14-39) at Chungnam National University Hospital Respectively.
Specifically, CRIF1 loxp / loxp mice (C57BL / 6 strain) inserted with a loxp site on the side of
The genotype of the selected mouse was confirmed by reverse transcription PCR (PCR), and it was confirmed that the CRIF1 gene was knocked out as shown in FIG. 1A (FIG. 1A). In addition, the selected CRIF1 homozygous knockout mice had a normal appearance and birth ratio at birth, but showed a tendency to decrease in body weight gain from about 10 weeks of age as compared to the control group (Fig. 1b), and the survival rate was also about 14 weeks (Fig. 1 (c)). The phenotype at 13 weeks of CRIF1 homozygous knockout mice is shown in Figure 1d (Figure 1d).
Experimental Example 1 Identification of physiological abnormal symptoms in muscle cell-specific CRIF1 homozygous knockout mice
Physiological abnormalities of muscle cell-specific CRIF1 homozygous knockout mice selected in Example 1 were confirmed as follows.
<1-1> Identification of muscle mass and muscle cells in muscle cell-specific CRIF1 homozygous knockout mice
(Gastrocnemius, Gas) muscles and tibialis anterior (TA) / long toes in 13 week old male CRIF1 homozygous knockout mice (CRIF1 Δsm) and control mice (CRIF f / f ) Extensor digitorum longus (EDL) was excised and muscle was identified. As a result, as shown in FIG. 2A, 13-week-old male CRIF1 homozygous knockout mice showed a decrease in muscle mass as compared with the control mice (FIG. 2a).
Weights and weights of organs and muscles were also measured in 8-week-old CRIF1 homozygous knockout mice and control mice. As a result, the weight of epididymal white adipose tissue (eWAT), long toe extensor (EDL), and gastrocnemius (Gas) was significantly decreased in the CRIF1 homozygous knockout mice at 8 weeks of age (Fig. 2A).
The long toe extensor was also stained with hematoxylin & eosin according to methods commonly known in the art. As a result, as shown in FIG. 2C, it was confirmed that the muscle bundle area of muscle was significantly decreased in 13-week-old CRIF1 homozygous knockout mice as compared with the control mice (FIG. 2C).
<1-2> Identification of mitochondrial activity in muscles of muscle cell-specific CRIF1 homozygous knockout mice
To confirm mitochondrial activity in the muscle of muscle cell specific CRIF1 homozygous knockout mice, we confirmed as follows.
Specifically, slides frozen in muscle tissue were stained with SDH reaction solution (0.2 M succinic acid, 0.2 M phosphate buffer, pH 7.4, 0.2% Nitor-BT solution, Sigma) for 30 min at 37 ° C. After that, the cells were fixed with 10% formalin solution, washed with 15% ethanol solution, sealed with glycerin gelatin solution, and analyzed by optical microscope.
As a result, the activity of succinic acid dehydrogenase was decreased in CRIF1 homozygous knockout mouse as shown in FIG. 3, and mitochondrial activity was decreased in muscle of CRIF1 homozygous knockout mouse (FIG. 3).
<1-3> Exercise ability of muscle-specific CRIF1 homozygous knockout mice
The athletic performance of 13-week-old male CRIF1 homozygous knockout mice was determined as follows.
Specifically, grip strength, mean latency and momentum were measured in 13 week old male control mice and CRIF1 homozygous knockout mice (N = 10). The grip strength was measured using a grip strength meter (Harvard Apparatus Ins, USA). The mean latency was evaluated in the absence of pre-treatment (Pre) and 3 days Min after three post-treatment sessions. The drop test was carried out for 2 days to 4 months (Mo) mice on a rota-rod (Panlab Rota-Rods, harvard apparatus Ins., USA) , The rod was operated for 5 minutes at a speed of 25 RPM, and the number of times the mouse fell off the rod was measured and evaluated.
As a result, as shown in FIG. 4, in all the measurement results, the exercise ability of the CIF1 homozygous knockout mice was significantly reduced as compared with the control mice, and it was confirmed that the exercise ability was remarkably decreased as the age of the mice was increased ).
Experimental Example 2 Identification of mitochondrial phenotype and mitochondrial respiratory chain complex in muscle cell-specific CRIF1 homozygous knockout mice
In order to confirm the abnormality of mitochondrial phenotype and respiratory chain complex in muscle cells of CRIF1 homozygous knockout mice, the following experiment was conducted.
Specifically, long-toe extensor muscle (EDL) muscles were separated from 3-week-old male control mice and CRIF1 homozygous knockout mice (N = 10) and mitochondria were observed with a mouse electron microscope (JEOL, Tokyo, Japan). As a result, abnormal mitochondria in which the mitochondrial crystal structure was destroyed in CRIF1 homozygous knockout mice were identified as shown in Fig. 4A (Fig. 4A).
Western blot analysis was performed using representative proteins involved in respiratory chain complexes in order to confirm the expression levels of proteins involved in the mitochondrial respiratory chain complex.
As a result, as shown in FIG. 4B, in CRIF1 homozygous knockout mouse, NDUFA9 (
In order to confirm the assembly status of mitochondrial respiratory chain complex, mitochondria were isolated and purified from long-toe extensor muscle (EDL) muscle by glucose density gradient centrifugation and analyzed by BN-PAGE (Blue native-polyacrylamide gel electrophoresis, Life Techonologies , USA). As a result, as shown in FIG. 4C, assembly of the complexes I and III was inhibited in the CRIF1 homozygous knockout mouse as compared with the control mice, and it was confirmed that the mitochondrial abnormality (Fig. 4C).
<110> Foundation of Research & Business of Chungnam National University <120> Sarconia model mice, preparation method thereof and screening method of sarconia using them <130> 2015P-05-025 <160> 1 <170> KoPatentin <210> 1 <211> 1734 <212> DNA <213> Mus musculus <400> 1 acagccaaga tggcggcgct cgcaatgcgg agtggctatc tcctgcggct ctctgtggct 60 ctgggtccca ggtcccgcag ctaccgtgcg cccccgcccc cgcgccgccg tcccgggccc 120 cactcgccag acccggagaa cctgctgacc ccgcgatggc agctaacgcc ccgctatgtg 180 gccaagcagt tcggacgaca tggcgccatc tccggggtgc ccccggcctc cctgtggccc 240 accccagagc agctgcgcga gttggaggcc gaggagcaag aatggtaccc gagcttagcg 300 accatgcaag agtcgctgcg cttgcagcag caggccttgg aggcaaggcg ccaagccagg 360 gagcagcgta tcgcagagtg catggccaag atgccacaaa tgattgaaaa ctggcgaaag 420 cagaagcgag aacgctggga gaaaattcag gctgacaagg agcggagggc ccggttacag 480 gctgaggccc aggaacgcct gggctaccac gtggacccaa ggagtgctcg cttccaggaa 540 ctattacagg acctagacaa gcagcaacga aagcgcctaa aggaagaaag acaacgacag 600 aagaaggagg cacgaattgc tgctatggcc tctgctgaag cccaggactc agcagtgtct 660 ggtgaaccca gctcctgaaa gctcccttcc caataaagcc agctgctgac aacccatatt 720 ctacacattc tccctcaagt tgtgacttcc tgggtcgccc ggctcattcc ccaacacctg 780 ggcgggagaa tccctccacc tggctgtgtt cacacctgga cactaggcca tgccatgaac 840 tggggctttg gggagagaga aaggggaatg gatgggcaaa taatgaagga gcagatggca 900 ggagaggagg aggcttctgt ttaccaacat taatctccag taattagcca attaccaggg 960 ggagtacagc caaacagact gcatgataga aggagcagca gtccttctag ggttgagctg 1020 aggcaggggc ccaagtttcc agagggagga gatgctgggg cccgtctgta aggctcctag 1080 gctcctcccc gtttctcagc atgcccactt cacctgtgcc cccggctagc tcagactcac 1140 aaacagctgt gagaagctcc agtaggcatg aaccattatt agggctcatt tttaaaactt 1200 tattcacgtc aaaaccttta tcagagatgt ggttctgtcc tggggaaggg gtggccttgg 1260 ccttcagaac tgatgataac cccttttcca ggctggaggc caaggccaga ctggggagaa 1320 ggcctttggc ttctaacata gacttactcc agatggaaag atctgggcca ctccaataga 1380 ggatcaaagc ttagggctcc agcttcccct catcatggtg gaggaaggag ggttgtcctt 1440 ggggatgctt cagggatggg gaagcaggca ggcgcccctc catctggctg cagggagctg 1500 ggacagaggc caagaggggc ccatctgtcg cctcatggtc ttgccaacaa cttttgagta 1560 ggctgggctg ttaagaggac ggggacagct gtctggccca ggtcttctcg gactgatatg 1620 ggaatggggg ccacttcagg agggtgggaa gctgggctgg gttttttttt gttgttgttt 1680 ttttaatggt ttttgatatt cttcctctta aacatgaata tatatatgtt atat 1734
Claims (6)
Obtaining a second generation mouse by crossing the heterozygous deficient mouse and CRIF loxp / loxp mouse; And
Selecting a CRIF1 homozygous knock out mouse having a genotype of CRIF1 loxp / loxp and MLC-cre M / + from said second generation mouse.
Measuring at least one of a muscle decline rate or an increase rate and a survival rate of the animal model after administration of the sample; And
And selecting a sample that improves at least one of a muscle reduction rate or an increase rate and a survival rate in comparison with a control group to which the sample is not administered.
Measuring the mobility of the animal model after administration of the sample; And
And selecting a sample that improves mobility in comparison with a control group to which the sample is not administered.
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| WO2018214823A1 (en) * | 2017-05-25 | 2018-11-29 | 朱献军 | METHOD FOR CONSTRUCTING MOUSE MODEL WITH CONDITIONAL KNOCKOUT OF TMEM30A GENE FROM PANCREATIC β CELL AND USE |
| KR20210109317A (en) | 2020-02-27 | 2021-09-06 | 서울대학교병원 | Sarconia and osteopenia model mice, preparation method thereof and screening method of using them |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2018214823A1 (en) * | 2017-05-25 | 2018-11-29 | 朱献军 | METHOD FOR CONSTRUCTING MOUSE MODEL WITH CONDITIONAL KNOCKOUT OF TMEM30A GENE FROM PANCREATIC β CELL AND USE |
| KR20210109317A (en) | 2020-02-27 | 2021-09-06 | 서울대학교병원 | Sarconia and osteopenia model mice, preparation method thereof and screening method of using them |
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