CN-121970721-A - Specific Foxp4 gene knockout mouse model, construction method and application thereof

Abstract

The invention belongs to the field of genetic engineering animal models, and particularly relates to a specific Foxp4 gene knockout mouse model, and a construction method and application thereof. The method utilizes a Cre-loxP system to obtain Foxp4-fl/fl and Albcre/+ mice by mating the Foxp4-fl/+ heterozygous mice with Alb-Cre mice, thereby constructing and obtaining a liver-specific Foxp4 knockout mouse model. The invention discovers the important role of Foxp4 in maintaining the liver steady-state function for the first time, and the deletion of the Foxp4 leads to phenotypes such as intrahepatic lipid accumulation, inflammatory cell infiltration, liver cell function injury, mild fibrosis, abnormal glycolipid metabolism and the like, is highly consistent with the early symptoms and molecular characteristics of NAFLD, and has important application value in researching the roles of Foxp4 genes in the occurrence and development mechanism of NAFLD and the maintenance of liver steady-state, developing NAFLD related therapeutic drugs and the like.

Inventors

• ZHAO BIN
• GUO LIWEI
• Peng Yachun

Assignees

• 浙江大学绍兴研究院

Dates

Publication Date

20260505

Application Date

20251230

Claims (8)

1. 1. A method for constructing a liver-specific Foxp4 gene knockout mouse model is characterized by specifically comprising the steps of utilizing a Cre-loxP system, and obtaining a Foxp4-fl/fl by mating Foxp4-Flox heterozygous mouse Foxp4-fl/+ with a liver tissue-specific Cre tool mouse Alb-Cre through a mating strategy, so as to construct and obtain the liver-specific Foxp4 knockout mouse model.
2. 2. The method of claim 1, wherein the mating policy comprises: selfing Foxp4-fl/+ mice to obtain F1 generation Foxp4-fl/fl mice, hybridizing Alb-Cre/+ mice with Foxp4-fl/+ mice at the same stage to obtain F1 generation Foxp4-fl/+; albcre/+ mice; S.2, hybridizing the F1 generation Foxp4-fl/fl mice constructed in the step S.1 with the F1 generation Foxp4-fl/+; albcre/+ mice to obtain F2 generation Foxp4-fl/fl and Foxp4-fl/fl mice; albcre/+ mice; s.3 crossing the Foxp4-fl/fl mice in step S.2 with Foxp4-fl/fl; albcre/+ mice to obtain F3 generation Foxp4-fl/fl and F3 generation Foxp4-fl/fl; albcre/+ mice.
3. 3. The method of claim 2, wherein the strain of mice is a C57BL/6J background.
4. 4. The method of claim 2, wherein steps s.1, s.2, s.3 further comprise PCR amplification of genomic DNA from the toe or tail of the mouse, agarose gel electrophoresis, and genotyping.
5. 5. The method of claim 2, wherein the primer pair used for PCR amplification to identify the genotype of liver-specific Foxp4 knockout mice is the primers shown in SEQ ID NO. 1 and SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.
6. 6. A liver-specific Foxp4 knockout mouse animal model obtained by the method of claim 1.
7. 7. The use of a liver-specific Foxp4 knockout mouse animal model obtained by the method of claim 1 in simulating early symptoms of non-alcoholic fatty liver disease, studying liver metabolic homeostasis and mechanisms of occurrence and development of non-alcoholic fatty liver disease, and for screening related drugs.
8. 8. Use of a liver-specific Foxp4 knockout mouse animal model obtained by the method of claim 1 in the preparation of a model animal for drug screening for the treatment of non-alcoholic fatty liver disease.

Description

Specific Foxp4 gene knockout mouse model, construction method and application thereof Technical Field The invention belongs to the field of genetic engineering animal models, and particularly relates to application of a Foxp4 gene conditional knockout mouse in simulating early symptoms of nonalcoholic fatty liver disease, researching liver metabolic homeostasis and screening related medicines. Background Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease with a global prevalence of about 30% and its disease spectrum encompasses a range of liver abnormalities such as simple steatosis, non-alcoholic steatohepatitis (Non-alcoholic steatohepatitis, NASH), liver necrosis, cirrhosis, hepatocellular carcinoma and liver failure. The main clinical manifestations of NAFLD are liver fat accumulation, insulin resistance and hepatocyte adiposity (> 5%). Recent studies have shown that development of NAFLD is associated with lipid accumulation, oxidative stress, endoplasmic reticulum stress and lipotoxicity. The theory of "multiple hit" suggests that under the genetic susceptibility background, the occurrence and development of NAFLD are driven by the combined actions of various factors such as insulin resistance, lipid metabolism disorder, intestinal flora disorder, bile acid metabolism abnormality, activation of the innate immune system, and hepatocyte death. Although several signaling pathways (e.g., AMPK, NF- κ B, PPARs, FXR, TLR) are known to be involved, the upstream key transcription factors that regulate these signaling pathways, especially those that play a decisive role in the early stages of disease, remain to be systematically explored. Clinically, the therapeutic strategy aiming at NAFLD mainly takes lifestyle (exercise and diet) intervention as a main part, and no effective medicines aiming at NAFLD are available at present, so that the pathogenesis of NAFLD is studied more deeply, and the searching of a new therapeutic target is vital. Animal models are an important basis for supporting research on NAFLD pathogenesis and development of new drugs. The current method for constructing NAFLD animal model mainly comprises diet induction, chemical induction, gene modification and the like. The diet induction model (such as high-fat diet, methionine-choline deficiency diet and western diet) can better simulate liver steatosis and inflammation under the background of human metabolism syndrome, but has long modeling period and large inter-individual difference, and is difficult to completely reproduce the complete pathological characteristics of the progress of human NAFLD to fibrosis. Although the chemical induction model (diet-chemical combination model) can accelerate fibrosis, the model is often accompanied by stronger hepatotoxicity, and the pathological process is greatly different from the natural course of human beings. Genetically modified models (e.g., ob/ob, db/db mice) have severe obesity and insulin resistance characteristics, and can rapidly develop significant liver steatosis, but are generally devoid of spontaneous inflammation and significant fibrosis. A common drawback of existing models is that they do not accurately mimic the precise pathological processes triggered by a loss of function of a specific gene in the liver, which is more closely related to the early human NAFLD. The transcription factor Foxp4 belongs to the fork box transcription factor P (Foxp) subfamily, and has dual functions in transcription control, and can be used as a transcription inhibitor or a transcription activator. Foxp4 has been found to be involved in processes such as spermatogonial stem cell proliferation, neural cell differentiation, and carcinogenesis. However, there is no report on the method of constructing a model of liver disease associated with Foxp4 knockout, nor is there any published evidence that Foxp4 is directly associated with NAFLD or liver homeostasis maintenance. Whereas studies have shown that FOXO4 dysfunction exacerbates the development of high-fat diet-induced NAFLD by up-regulating SPP1 expression. Therefore, constructing an animal model of NAFLD with liver specific Foxp4 knockout to study the occurrence and development mechanism of NAFLD and develop therapeutic drugs of NAFLD has great scientific value and clinical significance. Disclosure of Invention Aiming at the problems existing in the prior art, the invention aims at designing a specific Foxp4 gene knockout mouse model and a construction method and an application technical scheme thereof. The invention is realized by the following technical scheme: the first aspect of the invention provides a method for constructing a liver-specific Foxp4 gene knockout mouse model, which specifically comprises the steps of utilizing a Cre-loxP system to obtain a Foxp4-fl/+ mouse by crossing Foxp4-Flox heterozygous mice with liver tissue-specific Cre tool mice Alb-Cre through a mating strategy, and constructing a Albcre/+ mouse, th