Construction method and application of Slfn 4-deleted atherosclerosis model mouse
阅读说明：本技术 一种Slfn4缺失的动脉粥样硬化模型小鼠的构建方法及其应用 (Construction method and application of Slfn 4-deleted atherosclerosis model mouse ) 是由 郑前前 梁银明 卢燎勋 晁天柱 张黎琛 黄蓉 于 2020-06-12 设计创作，主要内容包括：本发明涉及一种Slfn4缺失的动脉粥样硬化模型小鼠的构建方法及其应用。本发明将Slfn4基因敲除通过CRISPR-Cas9系统引入ApoE基因缺失遗传背景的动脉粥样硬化小鼠,回交和杂交后进行基因型筛选,获得了双基因缺失的纯合子模型小鼠ApoE<Sup>–/–</Sup>Slfn4<Sup>–/–</Sup>。Slfn4缺失动脉粥样硬化模型小鼠(ApoE<Sup>–/–</Sup>Slfn4<Sup>–/–</Sup>)较ApoE<Sup>–/–</Sup>小鼠动脉粥样硬化斑块显著减少,症状显著减轻,表明Slfn4基因具有促进主动脉粥样硬化斑块形成的作用。针对Slfn4上述功能,Slfn4可以作为药物靶标筛选治疗动脉粥样硬化疾病的药物；Slfn4抑制剂能够用于制备治疗动脉粥样硬化疾病的药物。(The invention relates to a construction method and application of an atherosclerosis model mouse with a Slfn4 deficiency. According to the invention, Slfn4 gene is knocked out and ApoE gene deletion genetic background induced atherosclerosis mice through a CRISPR-Cas9 system, and genotype screening is carried out after backcross and hybridization to obtain a homozygote model mouse with double gene deletion ApoE –/– Slfn4 –/– . Slfn4 deficient atherosclerosis model mice (ApoE) –/– Slfn4 –/– ) Less than ApoE –/– The mice have obviously reduced atherosclerotic plaques and obviously relieved symptoms, and the Slfn4 gene has the function of promoting the formation of the aortic atherosclerotic plaques. Aiming at the functions of Slfn4, Slfn4 can be used as a drug target to screen drugs for treating atherosclerotic diseases; the Slfn4 inhibitor can be used for preparing medicines for treating atherosclerotic diseases.)
1. Slfn4 is absentThe method for constructing atherosclerosis model mouse is characterized in that ApoE is firstly used–/–Knocking out an Slfn4 gene of a mouse to obtain an F0 generation mouse; genotyping F0 mice and selecting F0 ApoE knocked out by Slfn4 gene–/–Background mice, and their administration with ApoE–/–Carrying out backcross on the mice to obtain F1 generation Slfn4 gene heterozygote mice; and finally selfing the F1 generation Slfn4 gene heterozygote mice to obtain F2 generation mice, and carrying out genotype identification and screening on the F2 generation mice to obtain a homozygous individual subjected to Slfn4 double allele knockout and ApoE double allele knockout, namely the Slfn4 gene knockout atherosclerosis model mouse.
2. The method for constructing an atherosclerosis model mouse with Slfn4 deficiency as claimed in claim 1, wherein the F0 generation mouse is obtained by applying ApoE using CRISPR/Cas9 technique–/–Mouse Slfn4 gene knockout.
3. The method for constructing an atherosclerosis model mouse with the deletion of Slfn4 as claimed in claim 2, wherein the obtaining of the F0 mouse generation comprises the following steps:
1) sgRNA selection targeting the mouse Slfn4 gene: in a CRISPR-Cas9 system, a sgRNA action site is positioned in a No. 2 exon region of a Slfn4 gene, and DNA target sequences of the sgRNA action site are respectively shown as follows:
Slfn4-sgRNA1：5’-AGGTGCAGGATACCAGGCAA GGG-3’；
Slfn4-sgRNA2：5’-AAGCCGAATCAGAGAGGTCC GGG-3’；
Slfn4-sgRNA3：5’-CTCAGTTGAACTGAAAGCAG CGG-3’；
2) designing a primer: designing 4 primers according to the sgRNA target sequence obtained in the step 1), wherein the primer sequences are as follows:
Slfn4-IVT-1：5’-TTCTAATACGACTCACTATAGGAGGTGCAGGATACCAGGCAAGTTTTAGAGCTAGA-3’；
Slfn4-IVT-2：5’-TTCTAATACGACTCACTATAGGAAGCCGAATCAGAGAGGTCCGTTTTAGAGCTAGA-3’；
Slfn4-IVT-3：5’-TTCTAATACGACTCACTATAGGCTCAGTTGAACTGAAAGCAGGTTTTAGAGCTAGA-3’；
Slfn4-IVT-4：5’-AAAAAAGCAC CGACTCGGTG CC-3’；
3) synthesis of double-stranded DNA fragments: pairing the Slfn4-IVT-1, Slfn4-IVT-2 and Slfn4-IVT-3 in the step 2) with Slfn4-IVT-4 respectively, carrying out polymerase chain reaction by taking a pX330 plasmid as a template, and purifying an obtained DNA amplification product;
4) obtaining sgRNA and Cas9 mRNA: respectively taking the double-stranded DNA fragments obtained in the step 3) as templates, and carrying out in vitro transcription and purification to obtain sgRNA1, sgRNA2 and sgRNA 3; carrying out in-vitro transcription and purification by taking AgeI single-enzyme digestion linearized pST1374-Cas9 plasmid as a template to obtain Cas9 mRNA;
5) obtaining Slfn 4-deleted F0-generation mice: proportionally injecting the 3 sgRNAs obtained in the step 4) and Cas9 mRNA into ApoE–/–Mouse sperms and fertilized egg cells generated by in-vitro fertilization of C57BL/6 mouse egg cells to obtain fertilized egg cells with a Slfn4 gene knockout, and transplanting the fertilized egg cells to a pseudopregnant female mouse, wherein the born mouse is the F0 generation mouse with the Slfn4 gene knockout.
4. The method for constructing an atherosclerosis model mouse with the Slfn4 deleted as claimed in claim 1, wherein the primers for genotyping the Slfn4 knockout mouse are as follows:
Slfn4-GeneTy-F：5’-TCGGAGAGAAGACTAGGGATTCA-3’；
Slfn4-GeneTy-R：5’-GCAGAAACAGTTTGAGGGAGG-3’。
5. the method for constructing an atherosclerosis model mouse with the deletion of Slfn4 as claimed in claim 4, wherein the Slfn4 genotype is analyzed by agarose gel electrophoresis.
6. The application of the Slfn 4-deleted atherosclerosis model mouse obtained by the construction method of claim 1 in researching the occurrence and development of atherosclerotic diseases.
7. The application of the Slfn 4-deleted atherosclerosis model mouse obtained by the construction method of claim 1 in researching the diagnosis and treatment of atherosclerosis.
Application of Slfn4 as a drug target in screening drugs for treating atherosclerotic diseases.
Application of an Slfn4 inhibitor in preparation of medicines for treating atherosclerotic diseases.
Technical Field
The invention relates to a construction method and application of an atherosclerosis model mouse with a Slfn4 deletion, belonging to the technical field of genetic engineering and genetic modification.
Background
Atherosclerosis is the main cause of vascular obstructive diseases, and cerebral infarction, myocardial infarction and the like caused by atherosclerosis are the leading cause of disability and lethality in China. The formation and development of atherosclerosis are closely related to mononuclear macrophages which phagocytose lipids to form foam cells, deposit on the blood vessel wall, release cytokines and recruit immune cells to form chronic inflammation, and the pathological basis of atherosclerosis is.
Chronic inflammation is a central factor leading to atherosclerosis. Immune cells continuously migrate to the blood vessel wall and accumulate together with lipids and the like to cause the formation of atherosclerotic plaques. Thrombosis after plaque rupture can lead to disabling and lethal consequences such as cerebral infarction, coronary atherosclerotic heart disease and the like. The immune cells in atherosclerotic plaques are mainly macrophages which are derived from monocytes in the circulating blood and phagocytose lipids to form foam cells. The effect of mononuclear macrophages on atherogenesis is reflected in two aspects: first, in the early stage of atherogenesis, monocytes in the blood pass through the endothelial space and differentiate into macrophages under the intima; macrophages bind to and engulf oxidized low density lipoprotein (Ox-LDL) through scavenger receptor CD36, tyrosine kinase Lyn is phosphorylated, and the phosphorylated Lyn cascade activates JNK1/2 or Vav and the like, thereby promoting Ox-LDL uptake, leading to intracellular lipid accumulation and forming foam cells. Second, after entering the intima, monocytes differentiate into two types of macrophages under the action of factors such as macrophage colony-stimulating factor (M-CSF): classical proinflammatory (M1) and anti-inflammatory (M2) macrophages, which focus on unstable and stable plaques, respectively, and exert a regulatory effect on the pathological course of atherosclerosis. Both are dynamic in the plaque, and macrophage numbers and typing affect the progression of the plaque. Therefore, the regulation and control measures for reducing macrophage lipid accumulation and inflammatory reaction have important significance for preventing and treating atherosclerotic cardiovascular diseases.
The myeloid differentiation factor Slfn4 is specifically and abundantly expressed in mononuclear macrophages, and the mononuclear macrophages have key effects on atherosclerosis, but the effects of Slfn4 on the differentiation of the mononuclear macrophages and the formation of foam cells under the hyperlipidemia condition are not studied.
Disclosure of Invention
The invention aims to provide a construction method of an atherosclerosis model mouse with a Slfn4 deletion function, explores the role of a Slfn4 gene in the occurrence and development of atherosclerosis diseases through the model, and provides a Slfn4 used as a target for screening a medicament for treating the atherosclerosis diseases and an application of a Slfn4 inhibitor in preparing a medicament for treating the atherosclerosis diseases.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for constructing an atherosclerosis model mouse with a Slfn4 deletion, which comprises the following steps: firstly ApoE–/–Knocking out an Slfn4 gene of a mouse to obtain an F0 generation mouse; genotyping F0 mice and selecting F0 ApoE knocked out by Slfn4 gene–/–Background mice, and their administration with ApoE–/–Carrying out backcross on the mice to obtain F1 generation Slfn4 gene heterozygote mice; and finally selfing the F1 generation Slfn4 gene heterozygote mice to obtain F2 generation mice, and carrying out genotype identification and screening on the F2 generation mice to obtain a homozygous individual subjected to Slfn4 double allele knockout and ApoE double allele knockout, namely the Slfn4 gene knockout atherosclerosis model mouse.
The method for obtaining the F0 mouse generation specifically comprises the following steps:
1) selection of sgrnas targeting the mouse Slfn4 gene: in a CRISPR-Cas9 system, a sgRNA action site is positioned in a No. 2 exon region of a Slfn4 gene, and DNA target sequences of the sgRNA action site are respectively shown as follows:
Slfn4-sgRNA1：5’-AGGTGCAGGATACCAGGCAA GGG-3’；
Slfn4-sgRNA2：5’-AAGCCGAATCAGAGAGGTCC GGG-3’；
Slfn4-sgRNA3：5’-CTCAGTTGAACTGAAAGCAG CGG-3’；
2) designing a primer: designing 3 primers according to the sgRNA target sequence obtained in the step 1), wherein the primer sequences are as follows:
Slfn4-IVT-1：5’-TTCTAATACGACTCACTATAGGAGGTGCAGGATACCA
GGCAAGTTTTAGAGCTAGA-3’；
Slfn4-IVT-2：5’-TTCTAATACGACTCACTATAGGAAGCCGAATCAGAGA
GGTCCGTTTTAGAGCTAGA-3’；
Slfn4-IVT-3：5’-TTCTAATACGACTCACTATAGGCTCAGTTGAACTGAA
AGCAGGTTTTAGAGCTAGA-3’；
Slfn4-IVT-4：5’-AAAAAAGCAC CGACTCGGTG CC-3’；
3) synthesis of double-stranded DNA fragments: pairing the Slfn4-IVT-1, Slfn4-IVT-2 and Slfn4-IVT-3 in the step 2) with Slfn4-IVT-4 respectively, carrying out polymerase chain reaction by taking a pX330 plasmid as a template, and purifying an obtained DNA amplification product.
4) Obtaining sgRNA and Cas9 mRNA: respectively taking the double-stranded DNA fragments obtained in the step 3) as templates, and carrying out in vitro transcription and purification to obtain sgRNA1, sgRNA2 and
5) obtaining an Slfn4 gene knockout F0 generation mouse: proportionally injecting the 3 sgRNAs obtained in the step 4) and Cas9 mRNA into ApoE–/–Mouse sperms and fertilized egg cells formed by in-vitro fertilization of C57BL/6 mouse egg cells to obtain fertilized egg cells with a Slfn4 gene knockout function, and transplanting the fertilized egg cells to a pseudopregnant female mouse, wherein the born mouse is the F0 generation mouse with the Slfn4 gene knockout function.
In the step 1), an MGI database (http:// www.informatics.jax.org /) is used for obtaining an Slfn4 gene sequence, and an online software CRISPOR is used for screening 3 specific sites with high specificity in an Slfn4 gene Exon2 section to serve as a target sequence of sgRNA.
In the step 4), the injection concentration of the Cas9 mRNA, the sgRNA1, the sgRNA2 and the sgRNA3 is 50-100 ng/mu L.
According to the invention, Slfn4 gene is knocked out and ApoE gene deletion genetic background induced atherosclerosis mice through a CRISPR-Cas9 system, and genotype screening is carried out after backcross and hybridization to obtain a homozygote model mouse with double gene deletion ApoE–/–Slfn4–/–. ApoE obtained by the construction method of the invention–/–Slfn4–/–The model mouse can normally breed and suckle, the immune system has no significant difference with a wild mouse, and sufficient and reliable ApoE (apoE) can be provided for the subsequent research of the function of Slfn4 in the generation and development of atherosclerotic diseases, the diagnosis and treatment of atherosclerosis and the like–/–Slfn4–/–Model mice homozygous model mice with double gene deletions.
The SLfn 4-deleted atherosclerosis model mouse obtained by the construction method provided by the invention lays a foundation for researching the function of the Slfn4 gene in the occurrence and development of atherosclerosis diseases. Specifically, an atherosclerosis model mouse with a Slfn4 deletion can be used for researching the role of Slfn4 in the formation and repair of atherosclerosis or a diagnosis and treatment method of atherosclerosis.
In order to clarify the relation between the Slfn4 gene and atherosclerotic diseases and how Slfn4 influences the occurrence and the development of atherosclerotic disease course, the invention carries out further experimental research on the obtained atherosclerosis model mice with the Slfn4 deletion:
for Slfn 4-deleted atherosclerosis model mice (genotype is ApoE)–/–Slfn4–/–) And control mice (genotype ApoE)–/–) After feeding with high-fat feed for 12 weeks, heart tissue was fixed with 4% paraformaldehyde overnight, and aortic root and main part were treatedAnd (5) carrying out oil red O staining analysis on the artery region, and carrying out statistics on the aortic root or aortic region plaque area of the two groups of mice. The results show that compared to control mice, in ApoE–/–Slfn4–/–In the model mice, atherosclerotic plaques are obviously reduced, and the symptoms of atherosclerosis are obviously relieved, which indicates that the Slfn4 deletion atherosclerosis model mice are successfully constructed, and the Slfn4 deletion slows the lesion degree of atherosclerosis.
The invention determines that Slfn4 has the effect of promoting the formation of atherosclerotic plaques, and is mainly embodied in that Slfn4 can promote the occurrence and development of atherosclerosis.
Aiming at the effects of Slfn4, the invention provides application of Slfn4 as a drug target in screening drugs for treating atherosclerotic diseases.
Aiming at the effects of Slfn4, the invention provides application of a Slfn4 inhibitor in preparing a medicament for treating atherosclerotic diseases. The Slfn4 inhibitor is preferably siRNA of Slfn4 gene, Slfn4 antibody or other inhibitors capable of inhibiting expression of Slfn 4.
The invention realizes the knockout of Slfn4 gene in an atherosclerosis mouse, and obtains an atherosclerosis model mouse which is deleted in Slfn4 and can normally survive. Has very important basic research and practical application value and also provides a new idea for developing a new scheme for treating atherosclerosis. The invention determines 3 specific targeting sites aiming at the mouse Slfn4 gene, and experimental data show that the specific sites have higher shearing efficiency; the large-fragment-deleted Slfn4 gene knockout atherosclerosis mouse is obtained, on one hand, the Slfn4 gene can completely lose functions, and on the other hand, the large-batch Slfn4 genotype detection of subsequent progeny mice is facilitated. The invention discloses a function of Slfn4 gene in the generation and development of atherosclerosis disease: the Slfn4 gene has the function of promoting the formation of atherosclerotic plaques. Aiming at the functions of Slfn4, Slfn4 can be used as a drug target to screen drugs for treating atherosclerotic diseases; the Slfn4 inhibitor can be used for preparing medicines for treating atherosclerotic diseases. The method opens a new direction for deeply developing the research of the atherosclerosis, provides more choices for the clinical treatment of the atherosclerosis, and has very important theoretical and practical significance.
Drawings
FIG. 1 is ApoE–/–A design scheme diagram of sgRNA targeted for Slfn4 gene knockout in genetically background mice;
FIG. 2 shows ApoE–/–Genetic background Slfn4 gene knockout F1 mouse fluorescence PCR genotype detection map;
FIG. 3 shows ApoE–/–Genetic background Slfn4 gene knock-out F2 mouse Sanger sequencing map;
FIG. 4 shows ApoE–/–Slfn4–/–Model mice and control APOE–/–Re-analyzing the cycle intermediate chart of the mice fed with the high-fat feed for 12 days;
FIG. 5 shows ApoE–/–Slfn4–/–Model mice and control APOE–/–Biochemical index detection map of serum after the mice are fed with high-fat feed for 12 weeks;
FIG. 6 shows ApoE–/–Slfn4–/–Model mice and control APOE–/–Oil red O and HE staining analysis images of aortic root cryosection after mice are fed with high-fat feed for 12 weeks;
FIG. 7 is ApoE–/–Slfn4–/–Model mice and control APOE–/–Aortic oil red O staining analysis profile after mice were fed high fat diet for 12 weeks.
Detailed Description
The present invention will be described in further detail with reference to specific examples. ApoE knockout mice (genotype APOE-/-) purchased from Jackson Laboratory, USA, suffer from atherosclerosis after being fed with high-fat feed for several weeks, and are the classical tool mice commonly used for studying atherosclerosis. The formula of the high-fat feed is referred to the research Diets company No. D12108C. Dissolving 0.5g of oil red O in 100mL of 100% isopropanol to prepare oil red O liquid, storing the oil red O liquid in a refrigerator at 4 ℃, diluting the liquid with distilled water according to a ratio of 3:2 to prepare oil red O working solution before use, and filtering the oil red O working solution, wherein the oil red O working solution is normally wine red and has no precipitate. pX330 plasmid DNA was purchased from Addgene; the pT7-3 XFlag-hCas 9 plasmid was given as a gift by the yellow army for professor. Other experimental articles are not described and are all commercial products.