CN110564701B

CN110564701B - Application of ring finger protein Rnf20 gene

Info

Publication number: CN110564701B
Application number: CN201910898943.3A
Authority: CN
Inventors: 王彦芳; 梁小娟; 陶聪; 王超; 王亚君
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2019-09-23
Filing date: 2019-09-23
Publication date: 2021-07-06
Anticipated expiration: 2039-09-23
Also published as: CN110564701A

Abstract

The invention provides application of a ring finger protein Rnf20 gene. The invention utilizes a Cre-loxp system and Rnf20^Flox/FloxMouse and Adiponectin-Cre⁺Mating mice, and successfully preparing an adipose tissue specific Rnf20 gene knockout mouse model. The invention uses fat specificity Rnf20 gene to knock out mice (Rnf 20)^Flox/Flox；Adiponectin‑Cre⁺) And control group mice (Rnf 20)^Flox/Flox) As an experimental object, the function of the Rnf20 gene in fat metabolism is researched through an age and high fat diet induced animal model, and the result shows that compared with a control group of mice, the fat metabolism disorder of the fat-specific Rnf20 gene knockout mice can simulate the pathogenesis process of human lipodystrophy. The model is an ideal model for researching fat malnutrition diseases and has important application value.

Description

Application of ring finger protein Rnf20 gene

Technical Field

The invention relates to the technical field of biology, in particular to application of a ring finger protein Rnf20 gene.

Background

White fat plays an important role in maintaining the metabolic balance of the body, and metabolic disorders caused by abnormal functions of white fat become important threats to human health. The dysfunction of fat storage can mainly cause two diseases of obesity and lipodystrophy. Lipodystrophy is mainly characterized by complete or partial loss of adipose tissue. Congenital lipodystrophy is a rare autosomal recessive disease, which is clinically manifested by almost complete loss of adipose tissues in all parts of the body, accompanied by metabolic abnormalities, such as insulin resistance, impaired glucose tolerance, fatty liver, dyslipidemia, serum leptin, and decreased adiponectin level, and thus adipose tissues are considered as one of the most important endocrine organs of animals.

Histone post-translational modification is an important mode of epigenetic modification, and has special functions. Histone post-translational modifications mainly include methylation, acetylation, phosphorylation, ubiquitination, SUMO, adenylation, ADP-ribosylation, and the like. The single ubiquitination of the 120 th amino acid of histone H2B, i.e., H2Bub, plays an important role in transcriptional elongation, and in DNA damage response, cell differentiation, cancer, and the like.

Ubiquitin is a highly conserved protein of 76 amino acids with a molecular weight of 8.5kDa, and is widely present in eukaryotes. The ubiquitination process is continuously catalyzed by three enzymes, including ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3.

There are only a few E1 enzymes, approximately 40E 2 enzymes, and 500E 3 ligases in humans. Although monoubiquitination of H2B was discovered as early as 1980, it was unclear, in the last 20 years, about the enzymes responsible for monoubiquitination of H2B. Until 2000, the E2 ubiquitin conjugating enzyme of H2Bub (Rad 6 in yeast) was not found. After 3 years, E3 ligase Bre1 was again found in Saccharomyces cerevisiae. Homologues of Bre1, RNF20 and RNF40, responsible for ubiquitinating the 120 th lysine of mammalian H2B histones, were subsequently identified in mice. Specific recognition of the target protein by E3 plays a decisive role in the ubiquitination process. In mammals, two copies of RNF20, each included, form a complex with RNF40, acting as the primary E3 ligase, which interacts with the E2 binding enzyme and then catalyzes the H2 Bub.

As mammals, mouse genetic manipulation and phenotypic analysis techniques are the most mature and dominate in the development of animal models for various diseases. However, for a long time, the choice of a mouse model of lipodystrophy has been very limited. Therefore, an animal model capable of simulating human lipodystrophy needs to be built urgently, the occurrence mechanism of the animal model is deeply researched, and a new idea is provided for treating diseases related to lipodystrophy.

Disclosure of Invention

The invention aims to fill the blank of research on establishment of an animal model knocked out in mature adipose tissues by the Rnf20 gene, and research the influence of RNF20 on fat development and metabolism at the level of animal individuals. The invention aims to provide a correlation between ubiquitin ligase RNF20 and lipodystrophy, provide a new application of a target gene Rnf20 for treating lipodystrophy, and further apply the Rnf20 gene to improvement of lipodystrophy and treatment of diseases related to lipodystrophy.

In order to achieve the purpose of the invention, in a first aspect, the invention provides any one of the following applications of the ring finger protein Rnf20 gene:

1) for regulating fat development and metabolism;

2) used for constructing a lipodystrophy animal model;

3) used for constructing a fatty liver animal model induced by high fat diet;

4) used for constructing an animal model of hyperinsulinemia;

5) preparing a kit for manufacturing a lipodystrophy animal model;

6) preparing a kit for manufacturing a high fat diet induced fatty liver animal model;

7) preparing a kit for manufacturing an animal model of hyperinsulinemia.

In the present invention, the animal is a vertebrate, preferably a mouse.

The ring finger protein Rnf20 gene is a gene coding the following protein (a) or (b):

(a) 1, a protein consisting of an amino acid sequence shown in SEQ ID NO;

(b) 1, protein which is derived from (a) and has the same function by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 1.

In a second aspect, the present invention provides a method of constructing a lipodystrophy animal model, the method comprising: by utilizing a genetic engineering means, site-directed mutagenesis or knockout is carried out on the ring finger protein Rnf20 gene, the expression of the Rnf20 gene is inhibited or reduced from the transcription or translation level, or the function of the gene is deleted, and the obtained transgenic animal is used as a lipodystrophy animal model.

In a third aspect, the invention provides a method for constructing a lipodystrophy mouse model, using Rnf20^Flox/Flox(Xu, Song et al.2016) mice and Adiponectin-Cre⁺(Tao, Wang et al.2019) mice to obtain Rnf20^Flox/+；Adiponectin-Cre⁺Then, the mixture is mixed with Rnf20^Flox/FloxHybridizing the mice to obtain a fat-specific Rnf20 knockout mouse (Rnf 20)^Flox/Flox；Adiponectin-Cre⁺) As a lipodystrophy mouse model.

In a fourth aspect, the present invention provides the use of an animal model or mouse model constructed according to the above method for screening a medicament for preventing, alleviating and/or treating lipodystrophy, obesity, fatty liver and hyperinsulinemia.

In a fifth aspect, the invention provides any one of the following applications of a ring finger protein Rnf20 gene inhibitor, a ring finger protein Rnf20 inhibitor, a ring finger protein Rnf20 gene targeting vector or an editing system of a targeted ring finger protein Rnf20 gene:

a) reducing deposition of animal fat;

b) resistance to high fat diet and age-induced obesity;

c) induction of plasma hyperinsulinemia;

d) used for constructing a lipodystrophy animal model;

e) used for constructing a high fat diet induced fatty liver animal model;

f) used for constructing an animal model of hyperinsulinemia;

g) preparing a kit for manufacturing a lipodystrophy animal model;

h) preparing a kit for manufacturing a high fat diet induced fatty liver animal model;

i) preparing a kit for manufacturing an animal model of hyperinsulinemia.

Wherein, the ring finger protein Rnf20 gene inhibitor is a substance capable of inhibiting the expression of Rnf20 gene from the transcription or translation level, and the inhibitor includes but is not limited to shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA, low molecular compounds, peptides, antibodies and the like.

Optionally, the editing system of the targeted ring finger protein Rnf20 gene is a CRISPR/Cas9 system for targeted editing of the Rnf20 gene.

In a sixth aspect, the invention provides any one of the following applications of a ring finger protein Rnf20 gene promoter, a ring finger protein Rnf20 activator, a ring finger protein Rnf20 gene overexpression vector or a targeted ring finger protein Rnf20 gene editing system:

i) promoting deposition of animal fat;

ii) for reducing liver lipid accumulation;

iii) use for improving plasma hyperinsulinemia

iv) for increasing serum leptin, adiponectin levels;

v) for the prevention, alleviation and/or treatment of obesity;

vi) for ameliorating lipodystrophy;

vii) for the treatment of disorders associated with disorders of fat metabolism.

In a seventh aspect, the invention provides any one of the following uses of the ring finger protein RNF 20:

(1) for the prevention, alleviation and/or treatment of obesity;

(2) for ameliorating lipodystrophy;

(3) for treating disorders associated with disorders of fat metabolism;

(4) for the preparation of a medicament or composition for the prevention, alleviation and/or treatment of obesity;

(5) for the preparation of a medicament or composition for ameliorating lipodystrophy;

(6) for the preparation of a medicament or composition for the treatment of a disorder associated with fat metabolism;

in an eighth aspect, the present invention provides a medicament or composition for preventing, alleviating and/or treating obesity, the active ingredient being the ring finger protein RNF 20.

In a ninth aspect, the present invention provides a medicament or composition for ameliorating lipodystrophy and/or treating a disease associated with lipodystrophy, the active ingredient being the ring finger protein RNF 20.

The invention utilizes a Cre-loxp system and Rnf20^Flox/Flox(Xu, Song et al.2016) mice and Adiponectin-Cre⁺Rnf20 obtained by hybridization of (Tao, Wang et al.2019) mice^Flox/+；Adiponectin-Cre⁺The mice are then incubated with Rnf20^Flox/FloxMouse hybridization to obtain Rnf20^Flox/Flox；Adiponectin-Cre⁺And (3) knocking out a mouse by using the fat specificity Rnf20 gene, and successfully preparing a mouse model of knocking out the fat tissue specificity Rnf20 gene. The knockout of the adipose tissue specific Rnf20 gene of the animal model causes the reduction of the adipose deposition of the animal model, can resist high fat diet and age-induced obesity, is accompanied by phenotypes such as severe hyperinsulinemia, serum leptin and adiponectin level reduction, and is a typical lipodystrophy mouse animal model. The model can be used as an animal model for researching the specific functions of the Rnf20 gene in fat development and metabolism; meanwhile, the method can also be used for researching the pathogenesis of metabolic diseases such as lipodystrophy and the like, screening candidate substances for improving or treating the diseases, and has important application value.

The invention also provides the function of the Rnf20 gene in metabolic diseases such as lipodystrophy and the like.

The invention uses fat specificity Rnf20 gene to knock out mice (Rnf 20)^Flox/Flox；Adiponectin-Cre⁺) And control group mice (Rnf 20)^Flox/Flox) As an experimental object, the function of the Rnf20 gene in adipose tissue is researched through an age and high fat diet induced animal model, and the result shows that compared with a control group of mice, the fat metabolism disorder of the fat specificity Rnf20 gene knockout mice can simulate the pathogenesis process of human lipodystrophy. The model is an ideal model for researching fat malnutrition diseases and has important application value.

The present invention demonstrates for the first time the essential role of adipose tissue RNF20 in maintaining normal fat deposition, metabolism and normal insulin levels.

Drawings

FIG. 1 shows the results of PCR identification of a mature adipose tissue Rnf20 knockout mouse model (ASKO mouse) in example 1 of the present invention. Wherein, A: rnf20 genotype PCR amplification; b: cre genotype PCR amplification. In the figure, +/+ denotes Rnf20^+/+F/+ denotes Rnf20^Flox/+F/F denotes Rnf20^Flox/FloxAnd M represents a DNA molecular weight standard.

FIG. 2 is a graph showing the reduction of fat deposition in ASKO mice in example 2 of the present invention. Wherein, A: nuclear magnetic resonance imaging results of mice at 2 months, 6 months and 1 year; p <0.05, p <0.01, p <0.001 indicates significant or very significant differences. B: two major white adipose tissue morphologies in Wild Type (WT) and ASKO mice at 2 months, 6 months, and 1 year; c: white adipose tissue weight of WT and ASKO mice at 2 months, 6 months and 1 year (WT corrected to 1 for control group). Results are expressed as mean ± sem, with × p <0.001 indicating very significant differences. D: epididymal fat H & E staining was performed in two mice at 2 months, 6 months and 1 year.

FIG. 3 is a graph showing obesity of ASKO mice resistant to a high fat diet in example 2 of the present invention. Wherein, A: high fat diet induced body weight gain curve of mice; b: high fat diet induced WT and ASKO mice (left), and morphology of subcutaneous fat and epididymal fat (right); c: high fat induced H & E staining of adipose tissue in mice for 16 weeks. And D, the proportion of each organ of the mouse to the body weight is induced by high fat. Results are expressed as mean ± sem, with p <0.05 and p <0.01 indicating significant and very significant.

FIG. 4 shows that the Rnf20 gene knockout in example 3 of the present invention results in high insulin content in the plasma of mice. Wherein A is adiponectin level, B is leptin level, and C is insulin level. Results are expressed as mean ± sem, with p <0.05 and p <0.001 indicating significant and very significant.

FIG. 5 shows that the Rnf20 gene knockout in example 4 of the present invention results in the formation of fatty liver in mice. Wherein, A: high fat induced 16 weeks mice liver tissue photographs; b: high fat induced liver tissue weight in 16 weeks mice; c: high fat induced Triglyceride (TG) content in mouse liver tissue for 16 weeks; d: high fat induced H & E staining of mouse liver tissue for 16 weeks. Results are expressed as mean ± sem, with p <0.05 and p <0.01 indicating significant and very significant.

Detailed Description

The invention provides application of an Rnf20 gene in constructing a lipodystrophy vertebrate model.

Preferably, the animal is a vertebrate, preferably a mouse.

The application specifically comprises the step of constructing a lipodystrophy animal model by specifically knocking out an Rnf20 gene in an animal body through fat specificity.

The invention provides a method for constructing a lipodystrophy animal model, which comprises the following steps: respectively selects Rnf20^Flox/Flox(Xu, Song et al.2016) mice and Adiponectin-Cre⁺(Tao, Wang et al.2019) mice are bred to obtain Rnf20^Flox/+；Adiponectin-Cre⁺The mouse is then treated with Rnf20^Flox/FloxMouse hybridization to obtain Rnf20^Flox/Flox；Adiponectin-Cre⁺Fat-specific Rnf20 knockout mice.

The established lipodystrophy animal model can be further developed into a high fat diet-induced fatty liver animal model or a hyperinsulinemia animal model.

The invention also provides application of the animal model in screening medicines for treating lipodystrophy, obesity, fatty liver and hyperinsulinemia.

The invention also provides application of the animal model in researching the function of RNF20 in fat development and metabolism.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 preparation of Rnf20 knockout mouse

The RNF20 inactivated mutant mouse can be obtained by inserting exogenous DNA fragments such as transposon or virus into an Rnf20 gene sequence, or changing the gene sequence or reducing the gene expression by adopting a chemical mutagenesis method such as ENU, a physical mutagenesis method such as X-ray, or a gene targeting method based on embryonic stem cells or CRISPR/Cas9 technology.

In this embodiment, an SPF stage Rnf20 is adopted^Flox/FloxMice (C57BL/6J background) and adipoetin-Cre⁺(C57BL/6J background) mice were mated to obtain Rnf20^Flox/+；Adiponectin-Cre⁺The mouse is then treated with Rnf20^Flox/FloxObtaining knockout mice by mouse hybridization (Rnf 20)^Flox/Flox；Adiponectin-Cre⁺ASKO mice) and littermate control mice (Rnf 20)^Flox/FloxWT mice).

Mice were housed in cages under the following conditions: standard commercial mouse chow was purchased from australian diet ltd of beijing, ca (4.5% fat, 4% cellulose, 21% protein, 1.404kcal/g), high fat chow was purchased from Research Diets (60% fat, 20% carbohydrate, 20% protein), illuminated alternately every 12 hours at 24 ± 2 ℃, humidity 40% -70%, mice were fed with free water. Male and female mice at 8 weeks of age were 1: and (5) mating in cages according to the proportion of 2.

Genotype identification was performed on two-week-old young mice (fig. 1), which were weaned, labeled, and split into cages three weeks later. Not specifically stated, male mice were used for all experiments. The animals used in the experiment are regulated by the ethical committee of experimental animals of the Beijing institute of zootechnics and veterinary research, China academy of agricultural sciences.

Knockout mice were initially screened by PCR detection techniques.

The method for extracting DNA from rat tail tissue comprises the following steps:

1) 2 weeks old mice were removed and 0.5cm rat tails removed with scissors and transferred to a 1.5mL centrifuge tube and labeled.

2) Add 350. mu.L of lysis solution and 50. mu.L of proteinase K stock solution for lysis in a 56 ℃ water bath overnight.

3) The next day the samples were centrifuged at 12000rpm for 10min at room temperature.

4) The supernatant was poured into a 1.5mL labeled centrifuge tube, 800. mu.L absolute ethanol was added, and the tube was covered and shaken to reveal a flocculent precipitate.

5) Centrifuge at 12000rpm for 10 minutes at 4 ℃.

6) Discarding the supernatant, standing upside down and absorbing water for 3-5min until ethanol volatilizes.

7) Adding 100 mu L of TE liquid, blowing, beating and dissolving, and freezing and storing at-20 ℃ for later use.

The genotype identification method comprises the following steps:

the primers used were as follows:

Rnf20^Flox/Floxmouse PCR detection primer (Rnf20 genotyping primer):

Genotyping-Rnf20-F：5′-GCTGTAAGAGTTCTTAATGTATG-3′

Genotyping-Rnf20-R：5′-GGCTTGTCACACAAGCATGAGCATC-3′

Adiponectin-Cre mouse PCR detection primer (Cre genotyping primer):

Genotyping-Cre-F：5′-GCCTGCATTACCGGTCGATGC-3′

Genotyping-Cre-R：5′-CAGGGTGTTATAAGCAATCCC-3′

the PCR amplification system is as follows: 2 XTaq Master Mix 10 uL, 10 uM upstream and downstream primers 0.5 uL, DNA template 2 uL, ddH₂O 7μL。

The PCR amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, for 32 cycles; extending for 5min at 72 ℃, and storing for later use at 4 ℃.

mu.L of PCR product was mixed with 1. mu.L of 5 XGelRed agarose electrophoresis sample buffer (containing nucleic acid dye), loaded on 1.5% agarose gel, electrophoresed at 120V for about 30min, and photographed by a gel imaging system. The result is shown in figure 1, and the identification result shows that the Rnf20 gene knockout mouse is successfully constructed.

Example 2 Rnf20 Gene knock-out results in reduced fat deposition

With the age of the Rnf20 knockout mouse, the body components of the 2-month, 6-month and 1-year mouse are detected by using a nuclear magnetic resonance imager. Fat and muscle ratios were calculated to show that Rnf20 knockout mice had significantly lower fat composition than wild type mice, while body weight and muscle composition were significantly higher than wild type mice (fig. 2, a, and fig. 3, a). Meanwhile, physiological dissection was performed on high-fat and age-advanced induced obese mice, and Rnf20 knockout mice were found to have severe loss of adipose tissue at different sites (fig. 2, B and C, and fig. 3, B and D), and to be accompanied by hypertrophy of other tissues such as liver (fig. 3, D, and fig. 4, a and B). H & E staining of adipose tissue revealed that Rnf20 knockout mice adipocytes were significantly smaller than wild-type mice (fig. 2, D, and fig. 3, C). The above results fully indicate that Rnf20 knockout mice have severe adipose tissue loss. The specific method comprises the following steps:

1. tissue sampling of mice

Mice were opened to the skin and subcutaneous membranes along the ventral midline, exposing visceral organs. Taking tissues such as fat, liver, heart, kidney, spleen, etc., immediately weighing after taking materials, freezing in liquid nitrogen, and then transferring and freezing at-80 ℃. In addition, tissues such as fat for morphological observation were left and fixed overnight with 4% paraformaldehyde.

2. Tissue processing and pathological staining related experiments

(1) Tissue dehydration, transparency, waxing

The fixed part of the tissue in 4% paraformaldehyde is cut into a marked embedding frame and washed for more than 30 minutes in small-flow running water. The corresponding program is set on the machine according to the following flow, and the dehydration is carried out: 75% alcohol (45 minutes) → 85% alcohol (45 minutes) → 95% alcohol (45 minutes) → anhydrous alcohol (1 hour); ② transparent: xylene (1 hour) → xylene (1 hour); ③ soaking wax (65 ℃): paraffin (1 hour) → paraffin (1 hour). After the tissue is washed, the embedding frame containing the tissue is loaded into a basket of the machine, and the program is started. After the above procedures are completed, the tissue embedding frame is taken out and sent to a pathology room for embedding tissues, and meanwhile, the machine is cleaned for standby.

(2) Tissue section

Sections were cut using a microtome (slice thickness 5 μm).

(3) Tissue hematoxylin-eosin (HE) staining

The tissue paraffin section was put into a 65 ℃ oven (30 minutes) → xylene (5 minutes, 3 times) → 100% alcohol (1 minute) → 90% alcohol (1 minute) → 70% alcohol (1 minute) → distilled water washing → hematoxylin (5 minutes) → tap water washing off loose color on the section → 1% hydrochloric acid alcohol (1 to 3 seconds) → tap water washing time → Scott blue promoting solution (sodium hydrogen carbonate 0.35g, magnesium sulfate 2g, distilled water 100mL) (1 minute) → tap water washing time → eosin (1 minute) → distilled water washing off loose color on the section → 70% alcohol at one time → 90% alcohol at one time → 100% alcohol (30 seconds, 3 times) → xylene (2 minutes, 3 times) → photographing and sealing the sheet when the xylene is not dried, and the like.

Example 3 Rnf20 Gene knock-out results in hyperinsulinemia

Insulin (Insulin) is a hormone that balances lipid metabolism and carbohydrate metabolism. Lipodystrophy patients often have symptoms associated with elevated insulin in their serum. In order to detect the insulin level in the Rnf20 knockout mouse, the concentration of the hormone in the serum of the knockout mouse is detected by an enzyme-linked immunosorbent assay (ELISA). The results found that serum insulin of Rnf20 knockout mice at 2 months old, 6 months old and 16 weeks after high fat induction was significantly higher than that of wild type mice (fig. 4, C). The experimental results show that knocking out the Rnf20 gene in mouse adipose tissue will result in a hyperinsulinemic phenotype. Meanwhile, we examined the contents of adiponectin and leptin in the serum and found that the contents of these two adipocytokines were significantly lower in ASKO mice than in WT mice at various developmental stages (fig. 4, a and B), and these results all indicate that ASKO mice have typical lipodystrophy symptoms.

Example 4 Rnf20 Gene knockout leads to fatty liver formation under high fat diet Induction conditions

After being induced for 16 weeks by high-fat diet, the liver tissue weight of the ASKO mice is significantly higher than that of the control group mice (figure 5, A and B), and the content of triglyceride in the liver of the ASKO mice is significantly higher than that of the control group mice (figure 5, C) determined by a tissue glycerol detection kit. H & E staining of liver tissue we found that there were significantly more lipid droplets in the liver cells of ASKO mice than in the control group (fig. 5, D). The results fully indicate that the Rnf20 knockout mouse has severe fatty liver under the induction of high-fat diet.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Reference documents:

[1]Tao,C.,Y.Wang,Y.Zhao,J.Pan,Y.Fan,X.Liang,C.Cao,J.Zhao,M.J.Petris,K.Li and Y.Wang(2019)."Adipocyte-specific disruption of ATPase copper transporting alpha in mice accelerates lipoatrophy."Diabetologia.

[2]Xu,Z.,Z.Song,G.Li,H.Tu,W.Liu,Y.Liu,P.Wang,Y.Wang,X.Cui,C.Liu,Y.Shang,D.G.de Rooij,F.Gao and W.Li(2016)."H2B ubiquitination regulates meiotic recombination by promoting chromatin relaxation."Nucleic Acids Res.

sequence listing

<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences

Application of <120> ring finger protein Rnf20 gene

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945 950 955 960

Ala Phe Gly Ala Asn Asp Phe His Arg Ile Tyr Ile Gly

965 970

Claims

1. A method for constructing a lipodystrophy mouse model, comprising using Rnf20^Flox/FloxMouse and adipoectin-Cre⁺Mouse hybridization to obtain Rnf20^Flox/+；Adiponectin-Cre⁺After mice, the mice are further treated with Rnf20^Flox/FloxMouse hybridization to obtain Rnf20^FloxFlox；Adiponectin-Cre⁺Fat-specific Rnf20 knockout mice serve as a lipodystrophy mouse model.

2. Use of a mouse model constructed according to the method of claim 1 for screening a medicament for preventing, alleviating and/or treating lipodystrophy, obesity, fatty liver and hyperinsulinemia.