A method for establishing a mouse non-alcoholic fatty liver disease model based on the autophagy-lysosome pathway

Abstract

The present invention provides a method for establishing a non-alcoholic fatty liver disease model in mice based on the autophagy-lysosome pathway, which belongs to the field of genetic engineering technology. The TFE3 gene in mouse liver cells is specifically knocked out by CRISPR / Cas9 gene editing technology and the chromosome site-specific recombinase system Alb-Cre-Loxp, and then a non-alcoholic fatty liver disease mouse model is constructed by feeding a high-fat diet / normal feed. The new mouse NAFLD model has the characteristics of stability, controllability and immediate effect. It provides scientific research conditions for disease research and drug development, and also provides new ideas for further improving the construction of NAFLD models and the creation of animal models of human diseases.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a method for establishing a mouse non-alcoholic fatty liver disease model based on the autophagy-lysosome pathway. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) refers to a clinical pathological syndrome characterized by excessive fat deposition in hepatocytes, mainly triglycerides (TG), excluding alcohol and other clear liver-damaging factors. It includes simple fatty liver, nonalcoholic steatohepatitis and related liver cirrhosis. With the global prevalence of obesity and its related metabolic syndrome, NAFLD has become the second largest liver disease after viral hepatitis in my country. Among obese and diabetic patients, the incidence of NAFLD is as high as 70%, of which 10% to 20% will develop into nonalcoholic steatohepatitis, and the incidence of liver cirrhosis in the latter is as high as 25% within 10 years. In the near future, the harm of NAFLD to human health will continue to increase. In-depth research on NAFLD is a hot topic and new challenge in the current medical field. Therefore, it is particularly important to establish animal disease models induced by different methods and factors for the study of the pathogenesis of NAFLD and the development of drug treatment.

[0003] An ideal animal model meets the following criteria: (1) the onset characteristics are similar to those of human fatty liver disease; (2) the disease has a certain development process; (3) the formation rate is high, the mortality rate is low, the controllability is strong, and the repeatability is good; (4) the modeling method is simple, easy to operate, and inexpensive. Currently, the commonly used NAFLD animal models mainly include malnutrition fatty liver models (induced by high-fat diet, high carbohydrates, and choline-methionine deficiency), drug-induced fatty liver models, and special strain fatty liver models (such as genetically obese Zucker rats and BHE rats). However, although the above models can simulate the lipid deposition environment of the liver in vivo, they all have certain defects. For example, the choline-methionine deficiency-induced model is a classic animal model of non-alcoholic fatty liver disease, which is simple to operate and takes a short time to model. However, the choline-methionine deficiency feed is expensive, and the human liver does not contain choline oxidase, so there is no choline deficiency problem. Therefore, the pathogenesis of this model is completely different from that of humans, and it lacks the insulin resistance of human NAFLD. The drug-induced fatty liver model has a short modeling time and a high formation rate, but it presents an acute pathological process, which is essentially different from the pathogenesis and pathophysiological changes of human fatty liver. Due to the high toxicity of drugs, the mortality rate of experimental animals is high, and it is toxic to experimental personnel. Special strains of fatty liver rats are a type of animal model that is artificially selected and can form fatty liver congenitally or spontaneously without the need for acquired medication or adjustment of dietary structure. However, it is mainly used for the study of fatty liver with metabolic syndrome, but not for the study of fatty liver caused by acquired malnutrition. In addition, the source of special strains of rats is relatively scarce, expensive, and has a high mortality rate, so it is only suitable for the study of specific pathological mechanisms. The fatty liver model induced by high-fat diet is the most commonly used modeling method at home and abroad because of its simple operation method, low price, good repeatability, and low mortality rate. However, due to the complexity of human body functions and racial differences in experimental animals, as well as the lack of relatively unified standards in terms of animal age, feed formula, modeling time and model judgment indicators, no experimental animal model can fully simulate the evolution process of human non-alcoholic fatty liver disease, cannot accurately reflect the nature of fatty liver disease, and is difficult to meet the scientific research needs of all fatty liver diseases. Therefore, it is relatively difficult to establish an animal model that can fully reflect human fatty liver. Summary of the invention

[0004] Based on this, the present invention achieves specific knockout of TFE3 in mouse liver cells through CRISPR / Cas9 gene editing technology and the chromosome site-specific recombinase system Alb-Cre-Loxp (Alb: Albumin albumin, generally believed to be specifically expressed only in liver cells), in order to construct a new NAFLD model in mice.

[0005] The technical solution adopted by the present invention is:

[0006] The present invention provides a method for establishing a non-alcoholic fatty liver disease model in mice based on the autophagy-lysosome pathway. The specific construction method is: using CRISPR / Cas9 gene editing technology to knock out the original gene TFE3; then using gene knockout technology, integrating the TFE3 gene with the Loxp segment into the original genome to obtain Loxp mice; finally, mating Alb-Cre+ tool mice with Loxp mice to obtain mice with Cre and Loxp genes, that is, conditional knockout mice.

[0007] A method for establishing a mouse non-alcoholic fatty liver disease model based on the autophagy-lysosome pathway specifically includes:

[0008] Step 1: Based on the TFE3 gene sequence, use sgRNA Designer software to predict potential sgRNA sequences and select appropriate targets;

[0009] Step 2: sgRNA activity screening, select active and highly specific sgRNA;

[0010] Step 3: Construct sgRNA expression vector and perform in vitro transcription of sgRNA using T7 in vitro transcription kit;

[0011] Step 4: Use SP6 transcription kit to perform in vitro transcription of Cas9 mRNA;

[0012] Step 5: Mix Cas9 mRNA and sgRNA and perform microinjection into fertilized eggs;

[0013] Step 6: The injected fertilized eggs are transplanted into pseudo-pregnant female mice for further development;

[0014] Step 7: After the offspring mice are born, perform genotyping analysis on them;

[0015] Step 8: Obtain F0 generation gene mutant mice by identifying the born mice, and design a mouse mating and breeding program based on the F0 generation gene mutant mice;

[0016] Step 9: Obtain stable genetic offspring through step 8, then design groups, feed them with high-fat diet and normal feed respectively, and compare and determine the feeding route, that is, the formation time.

[0017] Furthermore, the specific operations of step 5 are as follows:

[0018] Step 501: Preparation of embryo donor mice;

[0019] Step 502: Embryo collection;

[0020] Step 503: Use M2 to make a long droplet on a glass slide, and cover it with paraffin oil to make a micromanipulation droplet. Use an oviduct to transfer a set of cells to the manipulation droplet, and observe under a microscope to ensure that the injection needle tip is not blocked and the DNA flow is smooth.

[0021] Step 504: Observe the zygote under a high-power microscope, select a zygote with two pronuclei and good morphology, adjust the holding needle to a position adjacent to the zygote, and adjust the pressure to fix it with negative pressure, requiring that the pronucleus should be on the central axis of the holding needle;

[0022] Step 505: Hold the needle and apply a little negative pressure to ensure that the zygote is firmly fixed, fine-tune the focus to make the edge of the pronucleus clearest, and then move the injection needle to make the needle tip completely clear;

[0023] Step 506: Push the injection needle through the zona pellucida, enter the egg cytoplasm and slowly approach the pronucleus. If the pronucleus swells, it indicates that the injection is successful. At this time, immediately withdraw the injection needle;

[0024] Step 507: After all zygotes in the operation droplet are injected, they should be placed in KSOM and cultured at 37°C for a period of time for the next step of embryo transplantation.

[0025] Furthermore, the specific operations of step 501 are:

[0026] Inject 5.0 IU of pregnant mare serum gonadotropin into the peritoneal cavity of mature C57BL / 6 female mice, inject PMSG at 13:00-14:00 in the afternoon, and then inject 5.0 IU of human chorionic gonadotropin 48 hours later. Immediately put them in the cage with male mice. Check the vaginal plugs of the female mice in the morning of the next day. The female mice that have successfully mated will be used to recover the embryos.

[0027] Furthermore, the specific operations of step 502 are:

[0028] After killing the mouse with thrombus, the abdominal cavity was opened, the oviduct was carefully separated, and then the oviduct was placed in M2 culture medium containing hyaluronidase. Under the action of hyaluronidase, the zygote gradually shed the cumulus cells. The zygote was then washed once in M2 culture medium and transferred to pre-balanced KSOM droplets for culture before injection.

[0029] Furthermore, the mouse mating and breeding scheme in step 8 is as follows:

[0030] Step 801: After the F0 generation mice have reached sexual maturity, they are mated with mice with the same mutation type to obtain F1 generation homozygous Loxp+ / + mice with stable mutations;

[0031] Step 802: mate Loxp+ / + mice with Alb-Cre+ / + mice to obtain homozygous gene mutant mice Alb-Cre+ / +; after loxp+ / +, mate the homozygous gene mutant mice with another batch of gene mutant mice to obtain stable genetic offspring.

[0032] Furthermore, the specific operations of the grouping design in step 9 are as follows:

[0033] Step 901: 24 stable genetic offspring Alb-Cre+ / +;loxp+ / + mice, weighing 20 g, were divided into six groups, 4 mice in each group, and Cre- / -;loxp+ / + mice were used as the control group;

[0034] Step 902: The first three groups of the six groups of Alb-Cre+ / +;loxp+ / + mice were fed with normal feed, and the last three groups were fed with a high-fat diet. The mice were killed in batches at 4 weeks, 8 weeks and 12 weeks, respectively, and the levels of pathological and biochemical indicators were evaluated to identify the non-alcoholic fatty liver model.

[0035] Beneficial effects of the present invention:

[0036] The present invention combines the autophagy-lysosome pathway to participate in the regulation mechanism of NAFLD, and uses gene editing technology to construct a new mouse NAFLD model. Specifically, through CRISPR / Cas9 gene editing technology and chromosome site-specific recombinase system Alb-Cre-Loxp, the TFE3 gene in mouse liver cells is specifically knocked out, and then a non-alcoholic fatty liver mouse model is constructed by feeding a high-fat diet / normal feed. The new mouse NAFLD model established has the characteristics of stability, controllability and immediate effect. It provides scientific research conditions for disease research and drug development, and at the same time provides new ideas for further improving the construction of NAFLD models and the creation of animal models of human diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1Schematic diagram of TFE3 regulating lysosome formation and autophagy; A is the observation of changes in the autophagy-lysosome pathway in each group by immunofluorescence, B, C, and D are the relative quantification of autophagosomes, lysosomes, and autophagy-lysosomes in each group, respectively; LV-Vector: empty control group; LV-TFE3: TFE3 overexpression group; LV-shTFE3: negative shRNA control; LV-shTFE3: TFE3 silencing group. ***P<0.001vs LV-vector group; ##P<0.05vs LV-vector group.

[0039] Figure 2 The effect of TFE3 on lipid deposition in NAFLD; A is the observation of lipid deposition in each group by microscope, and B is the relative quantitative comparison of lipid content in each group; LV-Vector: empty control group; LV-TFE3: TFE3 overexpression group; LV-shTFE3: negative shRNA control; LV-shTFE3: TFE3 silencing group; Scrambled siRNA: transfection of negative control RNA; Atg5 siRNA: transfection of Atg5 siRNA to block autophagy. ***P<0.001, compared with LV-vector group when only negative control siRNA was transfected; #P<0.05, compared with LV-vector group when only negative control siRNA was transfected; $P<0.05, $$P<0.001, compared with the negative control group transfected with the same group name.

[0040] Figure 3 TFE3 affects NAFLD by regulating lipolysis mediated by the autophagy-lysosome pathway; A is the observation of autophagy-lysosome pathway-mediated lipid decomposition by immunofluorescence, and B is the relative quantitative comparison result; where LV-Vector: empty control group; LV-TFE3: TFE3 overexpression group; LV-shTFE3: negative shRNA control; LV-shTFE3: TFE3 silencing group; Scrambled siRNA: transfection of negative control RNA; Atg5 siRNA: transfection of Atg5 siRNA to block autophagy. ***P<0.001, compared with the LV-vector group when only negative control siRNA was transfected; ##P<0.01, compared with the LV-vector group when only negative control siRNA was transfected; $$$P<0.001, compared with the negative control siRNA transfected group of the same group.

[0041] Figure 4To silence TFE3 expression, accelerate the progression of NAFLD and even form a spontaneous NAFLD model; Ad-shScram: injection of negative control shRNA adenovirus; Ad-shTFE3: injection of shTFE3 adenovirus. HFD 2weeks: high-fat diet for 2 weeks; NCD 8weeks: normal diet for 8 weeks.

[0042] Figure 5 Flowchart for constructing conditional knockout mice.

[0043] Figure 6 Mating scheme for conditional knockout mice. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] With the in-depth study of NAFLD in recent years, new understandings have been gained about its pathogenesis and treatment concepts. Studies have confirmed that autophagy is involved in regulating the degradation of intracellular lipid droplets. The enhancement of autophagy can alleviate NAFLD, while the loss of autophagy-related genes can aggravate NAFLD. Therefore, it is of great significance to clarify the relationship between hepatic steatosis and autophagy and its regulatory genes from a genetic perspective for seeking new treatment methods for NAFLD. Transcription factor E3 (translocation factor E3, TFE3) is a member of the microphthalmia-associated transcription factor (MiTF) family. The characteristic structures of this family of transcription factors are helix-loop-helix (HLH) and leucine zipper (LZ). The latest studies have shown that TFE3 plays an important role in the generation of lysosomes and the occurrence of autophagy. Lysosomes have the functions of digestion and transportation in cells. Coordinated lysosomal expression and regulation (CLEAR) elements are commonly found in the promoter regions of genes related to lysosome formation. The CLEAR structure contains E-box repeat sequences, which are the main binding sites of the MiTF transcription factor family, including TFE3. Therefore, overexpression of TFE3 can enhance binding to CLEAR elements, regulate the expression of various lysosome-related genes, and promote the synthesis of lysosomes. Lysosomes are important factors in the occurrence of autophagy. When TFE3 is overexpressed, the level of the autophagy-lysosome pathway of cells is also significantly enhanced. At the same time, the level of the autophagy-lysosome pathway of cells is weakened as TFE3 expression is downregulated.

[0046] Previous studies have shown that TFE3 can regulate NAFLD through the autophagy-lysosome pathway. Specifically, overexpression of TFE3 (LV-TFE3) can significantly increase lipolysis mediated by the autophagy-lysosome pathway and alleviate NAFLD; conversely, silencing TFE3 (LV-shTFE3) reduces lipolysis mediated by the autophagy-lysosome pathway and aggravates NAFLD ( Figure 1-3 ). We injected TFE3 shRNA adenovirus (Ad-shTFE3) and control shRNA adenovirus (Ad-shScram) through the tail vein of mice, and gave them a high-fat diet and a normal diet, respectively. The results showed that after only 2 weeks of high-fat diet, the Ad-shTFE3 group had significant lipid deposition (usually 16 weeks of high-fat diet is required). After about 8 weeks of normal diet, the Ad-shTFE3 group also showed significant lipid deposition ( Figure 4). The above results indicate that silencing TFE3 expression can accelerate the progression of NAFLD and even form a spontaneous NAFLD model. However, TFE3 has been reported to play a role in cell growth, body development, differentiation and functional regulation. If the TFE3 gene is knocked out systemically, it will inevitably cause side effects and even affect the survival of the body. Therefore, we intend to establish a hepatocyte-specific TFE3 gene knockout mouse through gene editing to further construct a new NAFLD model.

[0047] In this example, CRISPR / Cas9 gene editing technology and the chromosome site-specific recombinase system Alb-Cre-Loxp (Alb: Albumin albumin, generally believed to be only specifically expressed in liver cells) were used to achieve specific knockout of TFE3 in mouse liver cells, in order to construct a new mouse NAFLD model ( Figure 5 ). This study combined the autophagy-lysosome pathway with the regulation mechanism of NAFLD, and used gene editing technology to actively explore the construction of a new mouse NAFLD model, in order to provide scientific research conditions for disease research and drug development, and at the same time provide new ideas for further improving the construction of NAFLD models and the creation of animal models of human diseases.

[0048] Preliminary research foundation

[0049] 1. TFE3 regulates lysosome formation and autophagy ( Figure 1 The main purpose is to illustrate that in the fatty liver cell model, the autophagy-lysosome pathway can be affected by enhancing and reducing TFE3 expression. In particular, reducing TFE3 expression and reducing the autophagy-lysosome pathway are the core ideas of this patent. Figure 1 Figure A is the observation of the changes in the autophagy-lysosome pathway in each group by immunofluorescence. Figure 1 B. Figure 1 C. Figure 1 D is the relative quantification of autophagosomes, lysosomes, and autophagolysosomes in each group. ): In the NAFLD cell model, TFE3 was overexpressed and silenced, and it was found that TFE3 overexpression significantly increased lysosome generation and autophagy; on the contrary, after silencing TFE3 expression, lysosome generation and autophagy were significantly reduced. The green GFP-LC3 dots represent autophagosomes, and the red Lyso-Tracker is a lysosome staining probe. The fusion of the two represents the occurrence of autophagolysosomes.

[0050] 2. Effect of TFE3 on lipid deposition in NAFLD Figure 2 A is to observe the lipid deposition of each group through a microscope. Figure 2B is a relative quantitative comparison of lipid content in each group): When Atg5 siRNA was not transfected (only negative control Scrambled siRNA was transfected), the hepatocyte Oil Red O staining and its 520nm absorbance semi-quantitatively showed that compared with LV-Vector, the intracellular lipid deposition in the LV-TFE3 group was significantly reduced; while the intracellular lipid deposition in the LV-shTFE3 group was increased compared with the control group LV-shScram group. The above results suggest that regulating TFE3 expression can affect lipid deposition in hepatocytes. When Atg5 siRNA was added to block the autophagy process, the effect of LV-TFE3 group in alleviating lipid deposition was greatly reduced, almost restored to the level of the corresponding control group. This suggests that the autophagy-lysosome pathway may play a key role in the regulation of lipid deposition by TFE3.

[0051] 3. TFE3 affects NAFLD by regulating lipolysis mediated by the autophagy-lysosomal pathway ( Figure 3 A is the observation of autophagy-lysosome pathway-mediated lipid degradation by immunofluorescence. Figure 3 B is the relative quantitative comparison result): We found that the LV-TFE3 group had a large number of fusion points (yellow) of the autophagosome marker LC3 dots (red) and lipids (green), which represented the "lipophagy" process mediated by the autophagy-lysosome pathway, which was involved in the decomposition of fat, and significantly reduced lipid deposition in the LV-TFE3 group. In contrast, the LV-shTFE3 group had almost no "lipophagy" effect due to the reduction of autophagy. When the autophagy key gene Atg5 siRNA was transfected, the large number of "lipophagy" dots in the LV-TFE3 group almost disappeared, and the intracellular lipid deposition increased significantly compared with the corresponding Scrambled siRNA transfection group. After transfection of Atg5 siRNA, the two control groups of LV-Vector and LV-shScram also had fewer "lipophagy" dots than the corresponding Scrambled siRNA transfection group due to the blockade of autophagy. The above results further suggest that the "lipophagy" effect mediated by the autophagy-lysosome pathway plays a key role in TFE3 regulating NAFLD.

[0052] A method for establishing a mouse non-alcoholic fatty liver disease model based on the autophagy-lysosome pathway, the specific operation is as follows:

[0053] Step 1: Use sgRNADesigner software to predict potential sgRNA sequences for the TFE3 gene sequence and select appropriate targets based on experimental requirements;

[0054] (1) Search the TFE3 gene sequence and copy the base sequence to the sgRNA prediction website gRNADesigner: http: / / portals.broadinstitute.org / gpp / public / analysis-tools / grna-design-v1 to predict potential sgRNA sequences. Next, select the sgRNA target based on the prediction score;

[0055] (2) Screening for gRNA activity: Clone 400bp (800bp in total) upstream and downstream fragments of the genomic target site corresponding to the selected gRNA into the pUCA (Luc) plasmid to cause premature termination of luciferase protein translation and express non-functional luciferase. At the same time, construct the sgRNA into the px459 plasmid and co-transfect 293T cells with pUCA. The Cas9 protein can cut the target site to form a complete luciferase coding sequence, thereby expressing functional luciferase. Using the Luciferase report kit, the activity of luciferase is positively correlated with the activity of CRISPR / Cas9, and the activity of sgRNA is indicated by detecting the expression of luciferase.

[0056] Step 2: sgRNA activity screening to select active and highly specific sgRNA

[0057] (1) After determining the 20bp target sequence (the genomic sequence should be 20bp+NGG), the Forward sequence: cacc-G+20bp target, the Reverse sequence: 20bp target reverse complement, 5' plus aaac;

[0058] (2) The forward and reverse bases of the synthesized sgRNA (molar concentration of 100 μM) are annealed to allow base pairing and form a double-stranded structure. The px330 plasmid is digested with BbsI, and the digested vector is recovered;

[0059] (3) Dilute the annealed and paired sgRNA bases with ultrapure water at 1:200 and connect them to the recovered vector;

[0060] (4) The ligation product is transformed and plated on an ampicillin-resistant solid culture plate. A single clone colony is picked, the plasmid is extracted after expansion culture, and the ligation vector is sequenced using the human U6 universal primer to detect whether the sgRNA base fragment is correctly inserted into the px330 plasmid.

[0061] Step 3: Construct sgRNA expression vector and perform in vitro transcription of sgRNA using T7 in vitro transcription kit

[0062] (1) Determine the 20 bp sgRNA target sequence, ligate it into BbsI-digested pGS3-T7-sgRNA as described above, sequence and select the correctly inserted plasmid;

[0063] (2) Linearization of pGS3-T7-sgRNA plasmid: 1 μg of pGS3-T7-sgRNA plasmid was digested with DraI and reacted at 37°C for 3 h;

[0064] (3) Using the correctly constructed pGS3-T7-sgRNA as a template, prepare an in vitro transcription reaction solution;

[0065] (4) The above solutions were evenly mixed and then slightly centrifuged to collect the transcription reaction solution at the bottom of the reaction tube and reacted at 42°C for 2 hours;

[0066] (5) Add 3 μl RNase-Free DNase I to the 20 μl reaction system, mix thoroughly, and incubate at 37°C for 30 min.

[0067] (6) Add 77 μl of RNase-free water to terminate the reaction;

[0068] (7) Purify the transcribed RNA using the phenol chloroform and isopropanol method;

[0069] (8) taking a portion of the reaction solution and performing 4% agarose gel electrophoresis to confirm the RNA product after in vitro transcription;

[0070] (9) Determine RNA concentration for microinjection;

[0071] Step 4: In vitro transcription of Cas9 mRNA using the SP6 transcription kit

[0072] (1) Linearization of pSP6-Cas9 plasmid: 5 μg of pSP6-Cas9 plasmid was digested with NotI and reacted at 37°C for 5 h;

[0073] (2) 0.8% or 1% agarose gel electrophoresis, followed by DNA gel purification and recovery, and concentration determination;

[0074] (3) Using purified DNA as template, prepare the reaction solution according to the instructions of the mMessage mMachine SP6 in vitro transcription kit;

[0075] (4) The above solutions were evenly mixed and then slightly centrifuged to collect the transcription reaction solution at the bottom of the reaction tube and reacted at 37°C for 2 h;

[0076] (5) Add 1 μl of TURBO DNase to the reaction solution, mix well and react at 37°C for 15 min;

[0077] (6) Add 30 μl of nuclease-free pure water and 30 μl of lithium chloride solution, mix the solution and freeze it at -20°C for 30 min;

[0078] (7) Centrifuge at 4°C, 14,000 g for 10 min to precipitate mRNA, carefully remove the supernatant, wash once with 500 μl of 70% ethanol, and centrifuge again at 4°C, 14,000 g for 10 min;

[0079] (8) Aspirate the supernatant ethanol solution, dry at room temperature for 5 min, and finally dissolve the mRNA precipitate in 20 μl of RNase-free pure water. Mix well and place on ice to measure the concentration. Finally, store at -80°C or -150°C.

[0080] Step 5: Mix Cas9 mRNA and sgRNA and perform microinjection into fertilized eggs

[0081] (1) Preparation of embryo donor mice: 5.0 IU of pregnant mare serum gonadotropin (PMSG) is injected intraperitoneally into mature C57BL / 6 female mice, usually at 13:00 to 14:00 in the afternoon. About 48 hours later, 5.0 IU of human chorionic gonadotropin (hCG) is injected again and the mice are immediately caged with male mice. The vaginal plugs of the female mice are checked in the morning of the next day. The female mice that have successfully mated will be used to recover the embryos.

[0082] (2) Embryo collection: After killing the mice with thrombi, open the abdominal cavity and carefully separate the oviduct. Then put the oviduct into M2 medium containing hyaluronidase. Under the action of hyaluronidase, the zygote gradually sheds the cumulus cells. Then the zygote is washed once in M2 medium and transferred to the pre-balanced KSOM microdroplets for culture and injection;

[0083] (3) Use M2 to make a long droplet on a glass slide, and then cover it with paraffin oil to make a micromanipulation droplet. Use an oviduct to transfer a group of cells to the manipulation droplet, and observe under a microscope to make sure that the injection needle tip is not blocked and the DNA flow is smooth;

[0084] (4) Observe the zygote under a high-power microscope and select the zygote with two pronuclei and good morphology. Adjust the holding needle to a position close to the zygote and adjust the pressure to fix it with negative pressure. The pronucleus should be as close to the center axis of the holding needle as possible;

[0085] (5) Hold the needle and apply a little negative pressure to ensure that the zygote is firmly fixed, and fine-tune the focus to make the edge of the pronucleus clearest. Then move the injection needle to make the needle tip completely clear.

[0086] (6) Push the injection needle through the zona pellucida, into the cytoplasm of the egg and slowly approach the pronucleus. If the pronucleus swells, the injection is successful. At this time, the injection needle should be withdrawn immediately.

[0087] (7) After all zygotes in the operating droplet have been injected, they should be placed in KSOM and cultured at 37°C for a period of time for the next embryo transfer. Pay attention and keep records.

[0088] Step 6: The injected fertilized eggs are transplanted into pseudo-pregnant female mice for further development

[0089] To ensure the smooth progress of embryo transplantation, the vagina is cleaned before transplantation. The curvature of the transplant tube is adjusted according to the curvature of the cervix under B-ultrasound. The transplant tube with an inner core is gently inserted into the internal os of the cervix. After a slight breakthrough, the inner core is removed and the inner tube with the embryo is inserted into the middle of the uterine cavity and left to stand for a while. The embryo transplantation is completed.

[0090] Step 7: After the offspring mice are born, perform genotyping analysis on them;

[0091] Step 8: Design of mouse mating and breeding program: By identifying the mice born, we can generally obtain F0 generation gene mutant mice. When the F0 generation mice reach sexual maturity, they are mated with mice with the same mutation type to obtain F1 generation homozygous Loxp+ / + mice with stable mutations. Figure 6 The mating scheme shown above obtains homozygous mutant mice Alb-Cre+ / +; loxp+ / +, and then the homozygous mutant mice are mated with another batch of mutant mice to obtain stable genetic offspring.

[0092] Step 9: Design grouping: 24 stable genetic offspring Alb-Cre+ / +;loxp+ / + mice, weighing about 20 g, were divided into six groups, with 4 mice in each group. Cre- / -;loxp+ / + mice were used as the control group. The first three groups of the six groups of Alb-Cre+ / +;loxp+ / + mice were fed with normal feed, and the last three groups were fed with a high-fat diet. The mice were killed in batches at 4 weeks, 8 weeks and 12 weeks, respectively, and the levels of pathological and biochemical indicators were evaluated. The NAFLD model was identified to explore the optimal feeding route and formation time.

[0093] In step 6, embryo transfer can also be performed in the following ways:

[0094] (1) Weigh the mouse and inject the anesthetic intraperitoneally. After anesthesia, place the mouse on a pad or paper to facilitate operation under a microscope. Pull out the ovary, fallopian tube and part of the uterine horn according to conventional surgical methods.

[0095] (2) Find the opening of the fallopian tube (fimbria) or the enlarged ampulla of the fallopian tube under a microscope and select these two sites as the entrance for transplantation. Adjust the position of the mouse and the fallopian tube, insert the transplantation needle into the fimbria of the fallopian tube, and gently blow the embryo into it. If bubbles are seen in the fallopian tube, it means that the transplantation is successful.

[0096] Embryo transplantation uses conventional techniques in the art, and other methods of embryo transplantation besides the two methods mentioned above are also feasible.

[0097] Silencing TFE3 expression accelerates the progression of NAFLD and even forms a spontaneous NAFLD model ( Figure 4 ): TFE3 shRNA adenovirus (Ad-shTFE3) and control shRNA adenovirus (Ad-shScram) were injected through the tail vein of mice, and high-fat diet and normal diet were given respectively. The results showed that after only 2 weeks of high-fat diet, the Ad-shTFE3 group had significant lipid deposition (usually 16 weeks of high-fat diet is required). After about 8 weeks of normal diet, the Ad-shTFE3 group also showed significant lipid deposition ( Figure 4 ).

[0098] Basic flow chart for the construction of conditional knockout mice ( Figure 5 ).

[0099] Schematic diagram of mouse breeding scheme ( Figure 6 ): Alb-Cre+ / -;loxp+ / + or Alb-Cre+ / +;loxp+ / + are the final hepatocyte TFE3 gene specific knockout mice.

Claims

1. A method for establishing a mouse non-alcoholic fatty liver disease model based on the autophagy-lysosome pathway, characterized in that: The specific construction method is as follows: using CRISPR / Cas9 gene editing technology to knock out the original gene TFE3; then using gene knock-in technology, the TFE3 gene with the Loxp fragment is integrated into the original genome to obtain the target Loxp mouse; finally, the Alb-Cre+ tool mouse is mated with the Loxp mouse to obtain mice with Cre and Loxp genes, which are conditional knockout mice; The specific implementation method is as follows: Step 1: Based on the TFE3 gene sequence, use sgRNADesigner software to predict potential sgRNA sequences and select appropriate targets; Step 2: sgRNA activity screening, select active and highly specific sgRNA; Step 3: Construct sgRNA expression vector and perform in vitro transcription of sgRNA using T7 in vitro transcription kit; Step 4: Use SP6 transcription kit to perform in vitro transcription of Cas9 mRNA; Step 5: Mix Cas9 mRNA and sgRNA and perform microinjection into fertilized eggs; Step 6: The injected fertilized eggs are transplanted into pseudo-pregnant female mice for further development; Step 7: After the offspring mice are born, perform genotyping analysis on them; Step 8: Obtain F0 generation gene mutant mice by identifying the born mice, and design a mouse mating and breeding program based on the F0 generation gene mutant mice; Step 9: Obtain stable genetic offspring through step 8, then design groups, feed them with high-fat diet and normal feed respectively, and compare and determine the feeding route, i.e., the formation time; The mouse mating and breeding scheme in step 8 is as follows: Step 801: After the F0 generation mice have reached sexual maturity, they are mated with mice with the same mutation type to obtain F1 generation homozygous Loxp+ / + mice with stable mutations; Step 802: mate Loxp+ / + mice with Alb-Cre+ / + mice to obtain homozygous gene mutant mice Alb-Cre+ / +; after loxp+ / +, mate the homozygous gene mutant mice with another batch of gene mutant mice to obtain stable genetic offspring.

2. The method for establishing a mouse non-alcoholic fatty liver disease model based on the autophagy-lysosome pathway according to claim 1, characterized in that: Step 5: Step 501: Preparation of embryo donor mice; Step 502: Embryo collection; Step 503: Use M2 to make a long droplet on a glass slide, and cover it with paraffin oil to make a micromanipulation droplet. Use an oviduct to transfer a set of cells to the manipulation droplet, and observe under a microscope to ensure that the injection needle tip is not blocked and the DNA flow is smooth. Step 504: Observe the zygote under a high-power microscope, select a zygote with two pronuclei and good morphology, adjust the holding needle to a position adjacent to the zygote, and adjust the pressure to fix it with negative pressure, requiring that the pronucleus should be on the central axis of the holding needle; Step 505: Hold the needle and apply a little negative pressure to ensure that the zygote is firmly fixed, fine-tune the focus to make the edge of the pronucleus clearest, and then move the injection needle to make the needle tip completely clear; Step 506: Push the injection needle through the zona pellucida, enter the egg cytoplasm and slowly approach the pronucleus. If the pronucleus swells, it indicates that the injection is successful. At this time, immediately withdraw the injection needle; Step 507: After all zygotes in the operation droplet are injected, they should be placed in KSOM and cultured at 37°C for a period of time for the next step of embryo transplantation.

3. The method for establishing a mouse non-alcoholic fatty liver disease model based on the autophagy-lysosome pathway according to claim 2, characterized in that: Specific operations of step 501: Inject 5.0 IU of pregnant mare serum gonadotropin into the peritoneal cavity of mature C57BL / 6 female mice, inject PMSG at 13:00-14:00 in the afternoon, and then inject 5.0 IU of human chorionic gonadotropin 48 hours later. Immediately put them in the cage with male mice. Check the vaginal plugs of the female mice in the morning of the next day. The female mice that have successfully mated will be used to recover the embryos.

4. The method for establishing a mouse non-alcoholic fatty liver disease model based on the autophagy-lysosome pathway according to claim 3, characterized in that: Specific operations of step 502: After killing the mouse with thrombus, the abdominal cavity was opened, the oviduct was carefully separated, and then the oviduct was placed in M2 culture medium containing hyaluronidase. Under the action of hyaluronidase, the zygote gradually shed the cumulus cells. The zygote was then washed once in M2 culture medium and transferred to pre-balanced KSOM droplets for culture before injection.

5. The method for establishing a mouse non-alcoholic fatty liver disease model based on the autophagy-lysosome pathway according to claim 4, characterized in that: The specific operations of group design in step 9 are as follows: Step 901: 24 stable genetic offspring Alb-Cre+ / +;loxp+ / + mice, weighing 20 g, were divided into six groups, 4 mice in each group, and Cre- / -;loxp+ / + mice were used as the control group; Step 902: The first three groups of the six groups of Alb-Cre+ / +;loxp+ / + mice were fed with normal feed, and the last three groups were fed with a high-fat diet. The mice were killed in batches at 4 weeks, 8 weeks and 12 weeks, respectively, and the levels of pathological and biochemical indicators were evaluated to identify the non-alcoholic fatty liver model.

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