CN-121991951-A - Preparation method and application of aortic aneurysm animal model

Abstract

The invention relates to the field of animal models, in particular to sgRNA for preparing an aortic aneurysm animal model and a preparation method thereof, wherein a TB structural domain of an exon 57 part of an FBN1 gene is knocked out on Bama miniature pig cells by a CRISPR/Cas9 gene editing method. The important structural domain of the FBN1 gene and the TB structural domain of the exon 57 part are screened out by deep analysis of the structure and the function of the FBN1 gene and by combining bioinformatics prediction and disease model related research. The Bama miniature pig subjected to specific gene editing prepared by the invention is expected to show unique value in medical research, disease model construction or specific livestock production application, and provides powerful support for research and development in related fields.

Inventors

• WU XIAO

Assignees

• 上海市农业科学院

Dates

Publication Date

20260508

Application Date

20251217

Claims (10)

1. 1. The sgRNA for preparing the aortic aneurysm animal model is characterized in that the nucleotide sequence is shown as SEQ ID NO.1 or SEQ ID NO. 2.
2. 2. A recombinant vector comprising the sgRNA of claim 1.
3. 3. The recombinant vector according to claim 2, wherein the recombinant vector comprises the sgRNA of claim 1 and a gene encoding a Cas9 enzyme.
4. 4. Use of the sgRNA of claim 1 for the preparation of an animal model of an aortic aneurysm.
5. 5. Use of the recombinant vector of claim 2 in the preparation of an aortic aneurysm animal model.
6. 6. The use according to claim 4 or 5, wherein the aortic aneurysm animal model is a gene-edited pamabroad pig with a deletion of the TB domain sequence in exon 57 of the FBN1 gene.
7. 7. The preparation method of the aortic aneurysm animal model is characterized in that a recombinant vector as claimed in claim 2 is adopted to knock out a TB structural domain of an exon 57 part of an FBN1 gene on Bama pig cells by a CRISPR/Cas9 gene editing method, and the obtained monoclonal is subjected to somatic cell transplantation technology to obtain a gene editing cloned pig, namely the aortic aneurysm animal model.
8. 8. The method of claim 7, wherein the Bama miniature pig cells are Bama miniature pig fibroblasts.
9. 9. The method of manufacturing according to claim 7, comprising the steps of: S1, co-transferring the sgRNA of claim 1 and Cas9 enzyme into Bama pig fibroblasts; S2, screening a heterozygous monoclonal cell strain with TB structural domain sequence deletion in a 57 th exon of the FBN1 gene; S3, obtaining the Bama pig with the TB structural domain sequence deletion in the 57 th exon of the FBN1 gene, namely an aortic aneurysm animal model, by a nuclear transfer technology from the monoclonal cell strain.
10. 10. Use of an aortic aneurysm animal model prepared by the preparation method according to any one of claims 7-9 for screening therapeutic agents and therapeutic methods for aortic aneurysm, and for studying pathogenesis of aortic aneurysm.

Description

Preparation method and application of aortic aneurysm animal model Technical Field The invention relates to the field of animal models, in particular to a preparation method and application of an aortic aneurysm animal model. Background In the prior art, the preparation method of the aortic aneurysm animal model mainly comprises an elastase (PPE) perfusion method, an angiotensin II (Ang II) induction method, a calcium chloride (CaCl 2) adventitia incubation method, a genetic engineering model and an operation forming method. The methods simulate the pathological process of the aortic aneurysm through different mechanisms, and provide an important experimental tool for research. However, these models also have some drawbacks and technical problems to be solved. First, the existing model is much different from human pathology. Most models (e.g., PPE or Ang II induced) are based on acute lesions, lacking the characteristic of slow progression of human aneurysms. Secondly, the model stability is poor, the mouse model is easy to rupture or retract the aneurysm, especially in young animals, the expansion of the aneurysm is unstable, and long-term observation and research are affected. In addition, the chest aortic aneurysm model has small quantity and large construction difficulty, and particularly, an effective means is still lacking in the aspect of simulating non-interlayer TAA. The surgical trauma is large, the death rate is high, particularly the thoracic aorta or a large animal model is complex in surgical operation, postoperative complications are numerous, and the success rate of the model is limited. At present, although a mouse model is edited by a thoracic aortic aneurysm gene, the circulatory hemodynamics of the mouse and a human body are greatly different, and a large animal model is required to be researched and validated in dynamic mechanism for researching and developing medicines and innovative surgical instruments for reversing pathological expansion of an ascending aorta. Furthermore, noninvasive therapies such as gene editing and the like must explore therapeutic efficacy and delivery routes through large animal models after verification in small animals, where they are hardly simulated. The surgically constructed model is generally devoid of the most essential wall pathology of clinically ascending aortic dilatation disease. Thus, studies conducted based on existing models, whether to explore the molecular mechanisms of the disease or evaluate the intervention effects of drugs (e.g., therapies aimed at reversing or delaying distension), may deviate from the true situation in the conclusion that core questions about the origin and genetic mechanisms of the disease cannot be resolved accurately. To overcome these limitations, it is desirable to develop animal models that more closely approximate the pathological features of humans, improving the stability and simulation of the model, in order to more accurately study the pathogenesis and treatment of aortic aneurysms. Disclosure of Invention The invention aims to provide sgRNA for preparing an aortic aneurysm animal model, application of the sgRNA and a preparation method of the aortic aneurysm animal model. The important structural domain of the FBN1 gene and the TB structural domain of the exon 57 part are screened out by deep analysis of the structure and the function of the FBN1 gene and by combining bioinformatics prediction and disease model related research. The TB structural domain of the exon 57 part of the FBN1 gene is knocked out on the Bama pig cells by a CRISPR/Cas9 gene editing method, and the obtained monoclonal is subjected to a somatic cell transplantation technology to obtain a gene editing cloned pig, namely the aortic aneurysm animal model. In a first aspect of the invention, there is provided an sgRNA for use in the preparation of an animal model of aortic aneurysm, the nucleotide sequence of which is shown in SEQ ID NO.1 or SEQ ID NO. 2. In a second aspect of the invention there is provided a recombinant vector comprising an sgRNA as described above. Further, the recombinant vector comprises the sgrnas and genes encoding Cas9 enzymes as described above. In a third aspect of the invention, there is provided the use of an sgRNA as described above for the preparation of an animal model of an aortic aneurysm. In a fourth aspect of the invention, there is provided the use of a recombinant vector as described above for the preparation of an animal model of an aortic aneurysm. Further, the aortic aneurysm animal model is a gene editing Bama pig with a deletion of TB domain sequence in exon 57 of the FBN1 gene. In a fifth aspect of the present invention, a preparation method of an aortic aneurysm animal model is provided, wherein a recombinant vector is adopted to knock out a TB structural domain of an exon 57 part of an FBN1 gene on Bama miniature pig cells by a CRISPR/Cas9 gene editing method, and the obtained monoclonal is subj