Application of Abcg4 gene in constructing mouse model of cerebral atherosclerosis with lung enlargement

Abstract

The present invention belongs to the field of animal genetic engineering and genetic modification, and specifically, it relates to the application of the Abcg4 gene in constructing a mouse model of cerebral atherosclerosis with lung enlargement. The application is specifically to obtain the mouse model of cerebral atherosclerosis with lung enlargement by knocking out the Abcg4 gene in the mouse. The mouse model of the present invention has a shorter time to see plaque formation, and cerebrovascular lesions are more obvious. It can be used as a drug screening platform to screen drugs that can target the treatment of cerebral atherosclerosis. The mouse model of the present invention not only has significant brain plaque formation, but also is accompanied by significant enlargement and weight gain of the lungs, which means that the function of macrophages is severely impaired. Compared with the model obtained by methods such as high-fat feed feeding, it has a wider range of applications.

Description

Technical Field

[0001] The invention belongs to the field of animal genetic engineering and genetic modification, and specifically relates to the application of Abcg4 gene in constructing a mouse model of cerebral atherosclerosis with lung enlargement. Background Art

[0002] Atherosclerosis (AS) is caused by damage to the intima of the artery, which leads to the accumulation of fat and fibrous substances in the innermost layer of the artery to form plaque-like substances, causing necrosis and softening of local tissues, forming a porridge-like substance and depositing on the surface of the intima, hence the name atherosclerosis. Over time, the fibrosis of atherosclerotic plaques gradually increases, and further accumulation of calcium minerals leads to narrowing of the blood vessel lumen, obstruction of blood flow, and even local blood supply deficiency or tissue necrosis.

[0003] At present, mice are the most commonly used species in atherosclerosis research, and many key features of the atherosclerosis process are similar to those of humans. The existing methods for producing animal models of atherosclerosis include high cholesterol and high fat diet feeding, immunological methods, catecholamine injection, homocysteine ​​injection, young rat method, and cholesterol-fat emulsion intravenous injection. The main methods for producing atherosclerosis models using gene knockout mice include knockout of low-density lipoprotein receptor LDL-R and knockout of apolipoprotein E (ApoE). There are no reports on the construction of atherosclerosis models by knocking out the Abcg4 gene.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] To fill the gap in the prior art, the present invention provides an application of the Abcg4 gene in constructing a mouse model of cerebral atherosclerosis with lung enlargement, characterized in that the application is specifically to obtain the mouse model of cerebral atherosclerosis with lung enlargement by knocking out the Abcg4 gene in the mouse.

[0006] Furthermore, the mice are C57BL / 6J mice.

[0007] Furthermore, the method for constructing the mouse model of cerebral atherosclerosis with lung enlargement comprises the following steps:

[0008] S1, using the wild-type C57BL / 6J mouse genome as a template, using primer pair I to amplify a fragment including exon 6 to part of exon 7 of the Abcg4 gene, recorded as fragment I;

[0009] The nucleotide sequence of the primer pair 1 is:

[0010] FI:5′-TGGTGGATGGTGCAGATGAC-3′;

[0011] RI:5′-AAATCGGGGTGGGTGCTTAGG3′;

[0012] The fragment containing exons 9 to 14 of the Abcg4 gene was amplified with primer pair II and was recorded as fragment II;

[0013] The nucleotide sequence of the primer pair II is:

[0014] FII:5′-AACTGACGAAGAAGCCGGAG3′;

[0015] RII: 5′-ATCAAAGAAGGACTGCCCCG3′;

[0016] S2, inserting fragment I into the eGFP coding region of the pCRII vector to obtain fusion fragment I, inserting fusion fragment I into the multi-cloning site A of the targeting vector OSDUPDEL, and inserting fragment II into the multi-cloning site B of the same targeting vector OSDUPDEL to obtain a targeted recombinant vector;

[0017] S3, after the above recombinant vector is injected into embryonic stem cells for homologous recombination, ES cell targeting products are obtained, positive clones of the ES cell targeting products are identified, and ES cells with positive knockout are screened;

[0018] S4, injecting the ES cells screened in S3 into C57BL / 6J mouse blastocysts and implanting them into pseudo-pregnant female mice to obtain heterozygous offspring mice with germline transmission ability;

[0019] S5, backcrossing the above heterozygous offspring mice with C57BL / 6J mice for N generations to produce a mouse strain with defects in important functional sites of the Abcg4 gene, and mating the heterozygotes with each other to generate Abcg4 gene-deficient mice, i.e., the mouse model of cerebral atherosclerosis with lung enlargement.

[0020] Furthermore, the nucleotide sequence of the primer pair II is:

[0021] FI:5′-TGGTGGATGGTGCAGATGAC-3′;

[0022] RI:5′-AAATCGGGGTGGGTGCTTAGG3′;

[0023] The nucleotide sequence of the primer pair II is:

[0024] FII:5′-AACTGACGAAGAAGCCGGAG3′;

[0025] RII: 5′-ATCAAAGAAGGACTGCCCCG3′.

[0026] Furthermore, in S5, N ≥ 16.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention uses gene-knockout mice as a specific animal model of cerebral atherosclerosis with lung enlargement, which is mainly prepared by reducing or completely knocking out the 4th member gene of the G subfamily of the ATP-binding cassette transporter superfamily (abbreviated as Abcg4 gene) in at least one tissue or at least one cell type of the mouse.

[0029] 2. The animal models constructed by the prior art cannot well reflect the conditions of cerebral vascular sclerosis and plaque formation, and the modeling time is relatively long. The mouse model of the present invention shows a shorter time for plaque formation, and the cerebrovascular lesions are more obvious. It can be used as a drug screening platform to screen drugs that can target the treatment of cerebral atherosclerosis.

[0030] 3. The mouse model of the present invention can simulate multiple disease characteristics. Not only is there significant brain plaque formation, but the lungs are also significantly enlarged and weighted, which means that the function of macrophages is severely impaired. Compared with models obtained by methods such as high-fat feed, it has a wider range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the strategy for constructing mice lacking important functional domains of the Abcg4 gene in Example 1.

[0032] Figure 2 These are H&E staining images of brain tissues of wild-type C57BL / 6J mice and Abcg4 gene-deficient mice, where a, b, and c are brain tissues of Abcg4 gene-deficient mice, and d, e, and f are brain tissues of wild-type C57BL / 6J mice.

[0033] Figure 3 Comparison of lung weights between wild-type C57BL / 6J mice and Abcg4 gene-deficient mice. DETAILED DESCRIPTION

[0034] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0035] Example 1: Construction of Abcg4 gene-deficient mice

[0036] See also Figure 1 The method for knocking out the mouse Abcg4 gene in this embodiment comprises the following steps:

[0037] S1, using the wild-type C57BL / 6J mouse genome as a template, using primer pair I to amplify a fragment including exon 6 to part of exon 7 of the Abcg4 gene, recorded as fragment I;

[0038] The nucleotide sequence of the primer pair 1 is:

[0039] FI: 5′-TGGTGGATGGTGCAGATGAC-3′ (as shown in SEQ ID NO. 1);

[0040] RI: 5′-AAATCGGGGTGGTGCTTAGG-3′ (as shown in SEQ ID NO. 2);

[0041] The fragment containing exons 9 to 14 of the Abcg4 gene was amplified with primer pair II and was recorded as fragment II;

[0042] The nucleotide sequence of the primer pair II is:

[0043] FII: 5′-AACTGACGAAGAAGCCGGAG-3′ (as shown in SEQ ID NO. 3);

[0044] RII: 5′-ATCAAAGAAGGACTGCCCCG-3′ (as shown in SEQ ID NO. 4);

[0045] The fragment containing exons 9 to 14 of the Abcg4 gene was amplified with primer pair II and was recorded as fragment II;

[0046] S2, inserting fragment I into the eGFP coding region of the pCRII vector to obtain fusion fragment I, inserting fusion fragment I into the multiple cloning site A (MCSA) of the targeting vector OSDUPDEL (OSDupDel Gene Targeting Vector-neo), and inserting fragment II into the multiple cloning site B (MCSB) of the same targeting vector OSDUPDEL to obtain a targeted recombinant vector;

[0047] The above PCR amplification utilized Roche's "Expanded Long Template PCR System" kit (Roche Applied Science, Indianapolis, IA, USA).

[0048] The program was as follows: initial denaturation at 94°C for 3 minutes; 10 cycles at 94°C for 15 seconds, 65°C for 30 seconds, and 68°C for 8 minutes; then 20 cycles at 94°C for 15 seconds, 65°C for 30 seconds, and 68°C for 8 minutes and 20 seconds; and finally extension at 68°C for 7 minutes.

[0049] S3, the above-mentioned targeted recombinant vector is injected into embryonic stem cells derived from C57BL / 6J mice for homologous recombination, and ES cell targeting products are obtained. The ES cell targeting products are positively cloned and identified, and ES cells with positive knockout are screened;

[0050] Positive ES cell genome identification method: The targeted recombinant vector was electroporated into C57B6 / J mouse embryonic stem cells, the transfected cells were positively selected with G418 and negatively selected with ganciclovir, and the surviving clones were screened by PCR and Southern blotting;

[0051] S4, injecting the ES cells screened in S3 into C57BL / 6J mouse blastocysts and implanting them into pseudo-pregnant female mice to obtain heterozygous offspring mice with germline transmission ability;

[0052] S5, the above heterozygous offspring mice were backcrossed with C57BL / 6J mice for 16 generations to produce a mouse strain with defects in important functional sites of the Abcg4 gene, and the heterozygotes were mated with each other to generate Abcg4 gene-deficient mice.

[0053] Example 2: Characterization of Abcg4 gene-deficient mice

[0054] 1. Analysis of cerebral atherosclerotic plaques

[0055] Experimental group: 6-month-old wild-type C57BL / 6J mice and Abcg4 gene-deficient mice of Example 1 were killed, and brain tissues were taken for frozen sections, stained with H&E staining, and photographed.

[0056] The results are as follows Figure 2 As shown, compared with wild-type mice, the blood vessels in the brains of Abcg4 gene-deficient mice showed cell proliferation and adhesion within the vessel wall (atherosclerotic plaque formation), and the vascular lumen became narrower.

[0057] 2. Lung volume and weight analysis

[0058] Six-month-old wild-type C57BL / 6J mice and the Abcg4 gene-deficient mice of Example 1 were killed, their lungs were removed, and the volume and weight of the lungs were measured and weighed.

[0059] The results are as follows Figure 3As shown, by measuring the lung volume and weight of these mice, it was found that the weight of Abcg4 gene-deficient mice, regardless of gender, was significantly increased compared with wild-type mice, meaning that the function of macrophages was severely impaired.

[0060] Example 3: Use of Abcg4 gene-deficient mice as a disease model of cerebral atherosclerosis with lung enlargement

[0061] The candidate drug is administered to the Abcg4 gene-deficient mice of Example 1. If the disease characteristics of cerebral atherosclerosis and lung enlargement in the Abcg4 gene-deficient mice are alleviated or reversed, it is proved that the candidate drug has therapeutic effect. Otherwise, it has no therapeutic effect.

[0062] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0063] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of Abcg4 gene in constructing a mouse model of cerebral atherosclerosis with lung enlargement, characterized in that: The application is specifically to obtain the mouse model of cerebral atherosclerosis with lung enlargement by knocking out the Abcg4 gene in mice, and the mice are C57BL / 6J mice.

2. The use according to claim 1, characterized in that: The method for constructing a mouse model of cerebral atherosclerosis with lung enlargement comprises the following steps: S1, using the wild-type C57BL / 6J mouse genome as a template, using primer pair I to amplify a fragment including exon 6 to part of exon 7 of the Abcg4 gene, recorded as fragment I; The fragment containing exons 9 to 14 of the Abcg4 gene was amplified with primer pair II and was recorded as fragment II; The nucleotide sequence of the primer pair 1 is: FI:5′-TGGTGGATGGTGCAGATGAC-3′; RI:5′-AAATCGGGGTGGGTGCTTAGG3′; The nucleotide sequence of the primer pair II is: FII:5′-AACTGACGAAGAAGCCGGAG3′; RII: 5′-ATCAAAGAAGGACTGCCCCG3′; S2, inserting fragment I into the eGFP coding region of the pCRII vector to obtain fusion fragment I, inserting fusion fragment I into the multi-cloning site A of the targeting vector OSDUPDEL, and inserting fragment II into the multi-cloning site B of the same targeting vector OSDUPDEL to obtain a targeted recombinant vector; S3, after the above recombinant vector is injected into embryonic stem cells for homologous recombination, ES cell targeting products are obtained, positive clones of the ES cell targeting products are identified, and ES cells with positive knockout are screened; S4, injecting the ES cells screened in S3 into C57BL / 6J mouse blastocysts and implanting them into pseudo-pregnant female mice to obtain heterozygous offspring mice with germline transmission ability; S5, backcrossing the above heterozygous offspring mice with C57BL / 6J mice for N generations to produce a mouse strain with defects in important functional sites of the Abcg4 gene, and mating the heterozygotes with each other to generate Abcg4 gene-deficient mice, i.e., the mouse model of cerebral atherosclerosis with lung enlargement.

3. The use according to claim 2, characterized in that: In S5, N≥16.

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