A method for constructing a mouse model of mitochondrial myopathy with a lars2 gene deletion and application thereof

Abstract

The application provides a method for constructing a mouse model of mitochondrial myopathy with a Lars2 gene deletion and application thereof, a first Lars2 flox / flox mouse is obtained by crossing an Acta1 ER‑Cre mouse with an Acta1 ER‑Cre -Lars2 flox / flox mouse; in the second step, the above mouse reaches 4 weeks of age, and tamoxifen 70-80 mg / kg is used for continuous intraperitoneal injection for 5 days; in the third step, 4 weeks later, tamoxifen 45-55 mg / kg is used for continuous intraperitoneal injection for 5 days; in the fourth step, the Acta1 ER‑Cre -Lars2 flox / flox mouse reaches 12-13 weeks of age, and skeletal muscle Lars2 protein deletion occurs, and skeletal muscle atrophy occurs obviously at 16 weeks of age, thereby obtaining a mouse model of mitochondrial myopathy with a Lars2 gene deletion.

Description

TECHNICAL FIELD

[0001] The application relates to a method for constructing a mouse model of mitochondrial myopathy with a Lars2 gene deletion and application thereof. BACKGROUND

[0002] Mitochondrial myopathy and mitochondrial encephalomyopathy are a group of multisystem diseases caused by abnormal structure and / or function of mitochondria, mainly involving brain and muscle. The disease usually occurs at the age of 20, and children and middle-aged people can also be affected. Recent studies have shown that the prevalence of mitochondrial myopathy caused by mitochondrial mtDNA mutation is 9.6 / 100,000 people, and the prevalence of mitochondrial myopathy caused by nuclear DNA mutation is 2.9 / 100,000 people. Mitochondrial myopathy is characterized by extreme fatigue of skeletal muscle, often feeling tired after light activity, and improving after rest. Muscle soreness and tenderness are common, but muscle atrophy is rare, and it is easy to be misdiagnosed as polymyositis, myasthenia gravis and progressive muscular dystrophy. The key manifestation is the dysfunction of the respiratory chain complex of mitochondria. In addition, 80% of mitochondrial myopathy patients have positive results in blood lactic acid and pyruvic acid minimum exercise test, that is, blood lactic acid and pyruvic acid cannot return to normal 10 minutes after exercise.

[0003] At present, mitochondrial myopathy can be divided into MELAS syndrome, MERRF syndrome, KSS syndrome, Pearson syndrome and other types according to its different clinical manifestations, auxiliary examination and histopathological characteristics. Although they all show dysfunction of mitochondrial respiratory chain complex, the pathogenesis is not the same and is not completely clear. Many studies have found that mutations in the mitochondrial aminoacyl-tRNA synthetase (AARS2) gene can cause mitochondrial myopathy. Among them, Christodoulou's research team reported that LARS2 gene mutation can cause mitochondrial myopathy with skeletal muscle respiratory chain complex I dysfunction, skeletal muscle type I fiber reduction and skeletal muscle atrophy as the main manifestations.

[0004] AARSs are enzymes that catalyze the formation of aminoacyl-tRNA complexes from various aminoacyls and corresponding tRNAs, thus playing a key role in protein synthesis. According to the functional characteristics and protein structure of AARSs, the family members can be divided into type I and type II AARSs. Type I AARSs contain two conserved amino acid sequences (HIGH and KMSKS) that are included in the "Rossmann fold" structural motif involved in ATP binding. In contrast, type II AARSs are composed of three structural motifs that link amino acids to the 3' OH of the terminal nucleotide of tRNA to form an active site. LARS1 belongs to type I AARSs, which has a Rossman fold, an inserted CP1 domain, a tRNA binding anticodon domain, and a C-terminal extension domain, catalyzing the synthesis of leucyl-tRNA complex. However, in recent years, studies have gradually found that the functions of AARS family members are not limited to the synthesis of aminoacyl-tRNA, and they also participate in the regulation of many important functions.

[0005] Yoon et al. suggested that LARS1 plays an important role in regulating leucine metabolism. And it can sense the intracellular leucine level and feedback to mTORC1, thereby regulating the protein synthesis, metabolism, autophagy, and cell proliferation of cells. Recent studies have found that LARS1 can positively regulate the differentiation of skeletal muscle by activating mTORC1. Son et al. found that LARS1 can negatively regulate skeletal muscle differentiation and skeletal muscle regeneration caused by injury. Whether positive or negative regulation, the above studies suggest that LARS1 plays an important role in skeletal muscle differentiation and muscle regeneration.

[0006] In 2000, Bullard's research team cloned and purified LARS2 protein. Similar to LARS1, LARS2 also contains characteristic type I AARS motifs and regions highly homologous to mitochondrial and bacterial LARS proteins of other species. Further analysis of the protein encoded by LARS2 cDNA showed that it contains 903 amino acids and a mitochondrial signal sequence composed of the first 21 amino acids. The monomer structure of the protein is the active state of the enzyme. It was found that human LARS2 can also catalyze the formation of aminoacyl-tRNA complex from E. coli tRNA.

[0007] In 2005, 't Hart found that LARS2 is a mitochondrial protein. LARS2 The gene may be one of the susceptibility genes for diabetes. Subsequent studies found that LARS2 gene mutations are associated with hearing loss and premature ovarian failure in Perrault syndrome, multisystemic dysfunction in infancy, and mitochondrial myopathy and related, in vitro studies have also found that LARS2 Gene mutations inhibit mitochondrial function in yeast.

[0008] The research team of Professor Zhu Yumin found that the deletion of LARS2 in ovarian cells leads to a decrease in mitochondrial membrane potential, an increase in oxygen free radicals, and causes mitochondrial dysfunction. In addition, the research of Professor Chu Yiming showed that the deletion of LARS2 in B lymphocytes leads to a decrease in the NAD + / NADH ratio in mitochondrial complex I, resulting in abnormal function of complex I and causing mitochondrial dysfunction. Our previously published research also showed that LARS2 deletion leads to mitochondrial fragmentation, increased mitochondrial oxygen free radicals, and inhibited oxidative phosphorylation in C2C12 myoblasts. However, the mechanism by which LARS2 causes mitochondrial dysfunction is still unknown. SUMMARY

[0009] The present application is directed to the above problems, and provides a method for constructing a mouse model of mitochondrial myopathy with LARS2 gene deletion and its application.

[0010] The application achieves the above-mentioned purpose by the following scheme: a method for constructing a mouse model of mitochondrial myopathy with LARS2 gene deletion,

[0011] First step: LARS2 flox / flox mice are crossed with Acta1 ER-Cre mice to obtain Acta1 ER-Cre - Lars2 flox / flox mice;

[0012] Second step: when the above-mentioned mice reach 4 weeks of age, tamoxifen 70-80 mg / kg is used for continuous intraperitoneal injection for 5 days;

[0013] Third step: after 4 weeks, tamoxifen 45-55 mg / kg is used for continuous intraperitoneal injection for 5 days again;

[0014] Fourth step: when the Acta1 ER-Cre - Lars2 flox / flox mice reach 12-13 weeks of age, skeletal muscle LARS2 protein deletion occurs, and at 16 weeks of age, skeletal muscle atrophy is obviously observed, thereby obtaining a mouse model of mitochondrial myopathy with LARS2 gene deletion.

[0015] Further, second step: when the above-mentioned mice reach 4 weeks of age, tamoxifen 75 mg / kg is used for continuous intraperitoneal injection for 5 days.

[0016] Further, third step: after 4 weeks, tamoxifen 50 mg / kg is used for continuous intraperitoneal injection for 5 days again.

[0017] A mouse model of mitochondrial myopathy with LARS2 gene deletion is used to study the role of LARS2 in the occurrence and development of mitochondrial myopathy.

[0018] The advantages of this invention are: it can obtain a stable mouse model of mitochondrial myopathy with Lars2 gene deletion; and it provides an application for studying the role of Lars2 in the occurrence and development of mitochondrial myopathy. Attached Figure Description

[0019] Figure 1 Lars2 flox / flox Mice and Acta1 ER-Cre Gene identification map of mice;

[0020] Figure 1 A is Lars2 flox / flox Mouse identification gel image, red bands represent homozygous Lars2 flox / flox ; Figure 1 B is Acta1 ER-Cre Gel prints of mice.

[0021] Figure 2 For Acta1 ER-Cre - Lars2 flox / flox Gross changes and skeletal muscle function assessment in mice;

[0022] Figure 2 A represents skeletal muscle LARS2 KO mice (Acta1) ER-Cre - Lars2 flox / flox Gross view and size comparison of tibialis anterior (TA) and gastrocnemius (GM) muscles with WT mice; Figure 2 B is a comparison chart of body weight, skeletal muscle mass, and fat mass; Figure 2 C is a graph comparing the exercise tolerance of mice on a treadmill. Figure 2 D is a comparison chart of the tension, tetanic contraction, and fatigue resistance of the tibialis anterior muscle under single stimulation.

[0023] Figure 3 For Acta1 ER-Cre - Lars2 flox / flox Histological evaluation of mouse skeleton;

[0024] Figure 3 A is Acta1 ER-Cre - Lars2 flox / flox Laminin staining and myotube area assessment of skeletal muscle in mice and control mice; Figure 3 B is Acta1 ER-Cre - Lars2 flox / flox SDH staining images of mouse and control mouse skeletons; Figure 3 C is a statistical graph showing the immunofluorescence staining and percentage content of different types of muscle fibers (I, IIa, IIb, IIx).

[0025] Figure 4 For Acta1 ER-Cre - Lars2flox / flox Figure 2 shows the evaluation of mitochondrial function and morphology in skeletal muscle of mice;

[0026] Figure 4 A is Acta1 ER-Cre - Lars2 flox / flox Figure 3 shows the structural changes in mitochondria of skeletal muscle of mice and control mice under electron microscopy; Figure 4 B is a graph showing the changes in mitochondrial complex activity and mitochondrial oxygen consumption in skeletal muscle of mice; Figure 4 C is a graph showing the changes in the expression levels of multiple proteins encoded by mtDNA in mitochondria of skeletal muscle of mice.

[0027] Figure 5 A is Acta1 ER-Cre - Lars2 flox / flox Figure 4 shows the flowchart of the crossbreeding of knockout mice and tamoxifen treatment of mice.

[0028] Figure 5 shows the Acta1 Cre A is Acta1 ER-Cre ; LARS2 fl / fl A is Lars2 flox / flox . DETAILED DESCRIPTION

[0029] The present application will be further described below in conjunction with specific examples:

[0030] The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available unless otherwise specified.

[0031] Example 1, refer to the attached Figures 1-5 A method for constructing a mouse model of mitochondrial myopathy with Lars2 gene deletion:

[0032] 1. Constructing a Lars2 conditional knockout mouse

[0033] The mouse Lars2 gene is located on chromosome 9. It has been found to contain 21 exons, of which the start codon is located in exon 2 and the stop codon is located in exon 21. We will select exon 4 (containing a 92 bp coding sequence) as the conditional knockout region. Using CRISPR-Cas gene editing technology, a loxP sequence is inserted at the 5' and 3' ends of exon 4 to obtain a Lars2 flox / flox mouse, which is a Lars2 conditional knockout mouse.

[0034] 2. Obtain a knockout mouse with skeletal muscle-specific knockout of Lars2

[0035] The above-mentioned Lars2 flox / floxThe mice were further compared with mice carrying cre (Acta1, a gene specifically expressed in mature skeletal muscle). ER-Cre Hybridization (provided by Cyagen Biotech) yielded Acta1 ER-Cre - Lars2 flox / flox Mice. At 4 weeks of age, the mice were administered tamoxifen (75 mg / kg) via intraperitoneal injection for 5 consecutive days. Four weeks later, they were again administered tamoxifen (50 mg / kg) via intraperitoneal injection for 5 consecutive days. These mice exhibited skeletal muscle Lars2 protein deficiency at 12-13 weeks of age, and significant skeletal muscle atrophy was observed at 16 weeks of age.

[0036] 3. Identification of gene-modified mice

[0037] Lars2 flox / flox Primers for mouse identification: F 5'-GTAGAGGTCGAGGACAGCTTATG-3', R 5'-GCAGAGCAGTAAGAGGCAAGTG-3'.

[0038] The mouse genotype identification was performed as follows: a mouse toe about 2 mm in length was cut off and immersed with the cut end facing down in lysis buffer (50 μl Buffer NP, 1 μl Foregene Protease Plus I), and placed in a 50°C oven for 30 min. The lysis products were stored at 4°C for later use.

[0039] Prepare the appropriate system in the PCR tube (12 μl 2×Rapid Taq Master Mix, 1 μl 10 μmol specific primers, 10 μl ddH2O). Add 1 μl of the prepared lysis mixture to the PCR system prepared above. Perform the PCR reaction according to the optimized PCR conditions. Detect the results by agarose gel electrophoresis at a constant voltage of 150V for 40 min.

[0040] Example 2, see attached document Figures 1-5 An application of a Lars2 gene-deficient mouse model of mitochondrial myopathy to study the role of Lars2 in the development and progression of mitochondrial myopathy:

[0041] 1. Assessment of skeletal muscle mass, exercise tolerance, and skeletal muscle tone in mice

[0042] ① Mouse skeletal muscle mass detection: Mouse skeletal muscle mass was detected using a small animal in vivo component analyzer, following its prescribed procedures. This instrument, based on low-resolution nuclear magnetic resonance (NMR), measures the lean tissue, adipose tissue, and water content in live small animals. After labeling, the mouse was fixed within the probe coil to prevent movement. The testing software was opened, calibrated, and the measurement began. Relevant data were recorded and statistically analyzed.

[0043] ②In vivo grip strength test: The mouse grip strength meter was carefully wiped with 75% alcohol in the biosafety cabinet, and the mouse grip strength meter was placed horizontally in the biosafety cabinet. The ultraviolet lamp was sterilized for 30 minutes. The fan was turned on 3 minutes before operation. Before the grip strength test, the mouse weight was measured and recorded. The mouse was placed on the grid of the grip strength meter, and the long axis of the mouse trunk was parallel to the long axis of the grip strength meter. The mouse tail was gently pulled, and the peak value of the mouse grip strength before escaping from the grid was recorded. Each mouse was measured 6 times, with 10 minutes rest each time. The grip strength of the mouse before and after modeling was recorded and statistically analyzed.

[0044] ③Mouse activity tolerance evaluation: We used Nanjing Calvin Technology Co., Ltd. KW-PT-S6 type 6 channel small animal treadmill to evaluate the mouse activity tolerance. Main parameter setting: acceleration 3 m / s, flat moving speed 18 m / min, inclination angle 5°, electric stimulation 0.7A, total number of stimulation 120 times. The total running time is 60 minutes. The judgment standard of mouse exhaustion is that the mouse cannot run and stays in the wire cage outside the flat plate to be stimulated by electricity for 120 times. After 120 times, the channel stops discharging and records the parameters such as exhaustion time, total running length, number of electric shocks, and first electric shock time.

[0045] ④Detection of tibialis anterior muscle function: Preload of skeletal muscle: isoflurane anesthetized mice, the limbs of the mice were fixed with adhesive tape, the abdominal side was upward, the skin of the right lower limb of the mouse was cut with tissue scissors, and the tibialis anterior muscle was exposed. The fascia was gently incised along the tibia with microforceps. The distal tendon of the tibialis anterior muscle was freed and disconnected, and the free end of the tendon was connected with the tension sensor by 3-0 surgical suture. The resting tension (preload) was adjusted to 3g. Detection of single contraction force: the common peroneal nerve was stimulated with pulse width 1ms and stimulation voltage 2-5V, and the maximum single contraction tension was recorded by the signal acquisition system. Detection of maximum isometric contraction force: under the same voltage, the initial delay was 0.1 seconds, the frequency increment mode was selected, and the maximum isometric contraction tension was recorded at an increment speed of 10Hz. Detection of fatigue index: after the skeletal muscle was fully rested for 2 minutes, the continuous stimulation mode was selected, the interval was 1ms, the stimulation voltage was the same as the maximum single contraction force, the stimulation frequency was the same as the maximum isometric contraction, and the continuous stimulation was 2 minutes. The time when the maximum contraction force decreased by 20% was the fatigue index.

[0046] 2, Morphological evaluation of mouse skeletal muscle

[0047] ①Evaluation of mouse skeletal muscle fiber cross-sectional area: The quadriceps femoris, tibialis anterior muscle, gastrocnemius muscle, and soleus muscle of the mouse were collected to make frozen sections. After being fixed with ice acetone and blocked with 10% BSA, laminin antibody was added for incubation at 4°C overnight. The next day, fluorescent secondary antibody was added for staining at room temperature for 1 hour. The muscle tube area was observed and counted under a fluorescence microscope.

[0048] ② Mouse skeletal muscle fiber type detection: Collect mouse quadriceps, tibialis anterior, gastrocnemius, soleus muscle to make frozen sections. After fixed with ice-acetone, 10% BSA blocking, add I, Ila, lib or IIx antibody 4℃ overnight incubation. Next day, add fluorescent secondary antibody room temperature staining for 1 hour. Observe under fluorescence microscope and count I, Ila, lib or IIx myotube number.

[0049] ③ SDH staining / NADH staining: Collect mouse quadriceps, tibialis anterior, gastrocnemius, soleus muscle to make frozen sections. SDH staining according to: Unfixed frozen sections are soaked in nitroblue tetrazolium chloride incubation solution, 37℃ incubation for 30 min. Distilled water is washed for 3 times, finally sealed with glycerol gelatin. NADH staining according to: Unfixed frozen sections are soaked in NADH incubation solution, put into NADH solution, 37℃ incubation for 30 min. Distilled water is washed for 3 times, finally sealed with glycerol gelatin. NADH solution formula: hydrochloric acid-Tris solution (PH 7.4) 10 ml + NBT powder 10 mg + NADH 8 mg, completely dissolved and filtered.

[0050] Count the number of deep purple, light purple and colorless muscle fibers under inverted phase contrast microscope and calculate the percentage to evaluate the mitochondrial SDH and NADH activity of skeletal muscle fibers.

[0051] 3. Mitochondrial function evaluation of mice

[0052] ① Use OROBOROS to detect skeletal muscle mitochondrial complex activity. Collect gastrocnemius muscle and weigh, take 100 mg of skeletal muscle. Use Dounce type tissue grinder to grind, extract mitochondria according to mitochondrial extraction kit. Resuspend 50 μl of mitochondrial storage solution, and take the same amount of mitochondria for standby after quantification by BCA. Mitochondrial complex activity is detected by OROBOROS: After oxygen tank of OROBOROS is calibrated, add 5 μl of the above mitochondrial suspension. Sequentially add 20 μl of pyruvate-malate-glutamate complex solution (Pyruvate-Malate-Glutamate, PMG), 5 μl of ADP, 5 μl of cytochrome c, 1 μl of rotenone, 20 μl of succinate (Succinate, Succ), 1 μl of antimycin A (Antimycin A, AA), 5 μl of ascorbate sodium salt (Ascorbate sodium salt, AS) + 5 μl of TMPD, 50 μl of azide (Azide, AZD), and record the oxygen consumption curve.

[0053] ② Collect soleus muscle and fix with 4% glutaraldehyde overnight. Wash three times with PBS, then fix with 1% osmium tetroxide for 2 hours. Wash three times with deionized water, then dehydrate sequentially with 50%, 70%, 80%, 90%, 100% ethanol, and 100% acetone, and coat with epoxy resin to form blocks. Section using an ultramicrotome (50 nm), stain with lead citrate and uranium acetate, and observe mitochondrial morphology and size under a transmission electron microscope. Randomly observe 1000 mitochondria, count the number of mitochondria, the percentage of mitochondria with abnormal structures, and statistically analyze mitochondrial size.

[0054] Example 3, see attached document Figure 1 Identification results of gene knockout mice: WT, Lars2 flox / flox Lars2 flox / - and Acta1 ER-Cre As indicated in the figure;

[0055] A. Lars2 flox / flox Mouse identification gel image, red bands represent homozygous Lars2 flox / flox B. Acta1 ER-Cre Gel prints of mice.

[0056] Example 4, see attached document Figure 2 Acta1 ER-Cre - Lars2 flox / flox Gross changes and skeletal muscle function assessment in mice;

[0057] Acta1 ER-Cre -Lars2 flox / flox Gene knockout mice were induced to knock out the LARS2 gene in mature skeletal muscle using tamoxifen. Skeletal muscle atrophy was observed in mice 16 weeks of age after tamoxifen injection. Figure 2 A), weight loss ( Figure 2 B). In vivo component analysis of small animals showed no significant decrease in adipose tissue mass in mice, while skeletal muscle mass was significantly reduced. Figure 2 B). Treadmill testing in small animals indicated decreased activity tolerance in mice. Figure 2 C). In vivo tibialis anterior muscle tension test indicated Acta1. ER-Cre -Lars2 flox / flox Knockout mice showed significantly reduced skeletal muscle tensile strength and fatigue resistance. Figure 2 D).

[0058] Example 5, see attached document Figure 3 Acta1 ER-Cre - Lars2 flox / flox Histological evaluation of mouse skeletal tissue indicated significant skeletal muscle atrophy;

[0059] By histological analysis of mouse skeletal muscle frozen sections, it was found that the cross-sectional area of skeletal muscle myotubes of LARS2 knockout mice (concentrated in 400-1000 μm 2 ) was significantly lower than that of control mice in the same cage (concentrated in 1000-1800 μm 2 ), suggesting skeletal muscle atrophy Figure 3 SDH staining showed that the mitochondrial SDH activity was not significantly reduced Figure 3 B). Acta1 ER-Cre -Lars2 flox / flox knockout mice showed significant changes in skeletal muscle fiber types, with a decrease in type I fibers and an increase in type II fibers, especially type IIa and type IIx Figure 3 C).

[0060] Example 6, refer to the attached Figure 4 , Acta1 ER-Cre -Lars2 flox / flox mouse skeletal muscle mitochondrial function and morphological evaluation;

[0061] Electron microscopy of skeletal muscle mitochondria showed that LARS2 deletion in skeletal muscle resulted in a large number of vacuolated mitochondria in skeletal muscle Figure 4 A). Isolated mouse gastrocnemius mitochondria were further detected by using OROBOROS, and it was found that the mitochondrial OCR and each complex function were decreased Figure 4 B), since LARS2 is involved in mtDNA translation, we detected the changes of 13 proteins encoded by mtDNA in mitochondria by protein electrophoresis. LARS2 deletion significantly down-regulated mitochondrial COX2 and ND6 expression Figure 4 C). The above results suggest that mitochondrial structure and function are abnormal.

[0062] Although the present application has been described by referring to preferred embodiments, it should be understood by those skilled in the art that various changes in form and details can be made within the scope of the claims without being limited to the description of the above embodiments.

Claims

1. A method for constructing a mouse model of mitochondrial myopathy with Lars2 gene deletion, characterized in that: Step 1: Lars2 flox / flox Mice via Acta1 ER-Cre Mouse hybridization yielded Acta1 ER-Cre - Lars2 flox / flox Mice; Step 2: When the mice reached 4 weeks of age, they were given tamoxifen 75mg / kg via intraperitoneal injection for 5 consecutive days. Step 3: Four weeks later, administer tamoxifen 50mg / kg via intraperitoneal injection for 5 consecutive days. Step 4: Acta1 ER-Cre - Lars2 flox / flox Mice showed Lars2 protein deficiency in skeletal muscle at 12-13 weeks of age, and skeletal muscle atrophy was obvious at 16 weeks of age, thus obtaining a mouse model of mitochondrial myopathy with Lars2 gene deletion.

2. The application of a Lars2 gene-deficient mouse model of mitochondrial myopathy as described in claim 1 for studying the role of Lars2 in the development and progression of mitochondrial myopathy.

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