Preparation method and application of BMPR2 gene mutant rat

A gene and mutation site technology, which is applied in the field of preparation of BMPR2 gene mutant rats, can solve the problem that the BMPR2 missense mutation cannot be reflected, the disease process and disease phenotype of pulmonary arterial hypertension cannot be truly simulated, and the clinical pathology of patients cannot be well simulated. phenotype and other issues, to achieve the effect of stable disease phenotype

Pending Publication Date: 2019-01-25
FUWAI HOSPITAL CHINESE ACAD OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE +1
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Problems solved by technology

[0004] 1) Currently commonly used rat models of pulmonary hypertension, whether monocrotaline (MCT) and hypoxia-induced pulmonary hypertension, or hypoxia combined with VEGF inhibitor SU5416-induced pulmonary hypertension, are all induced by specific environmental or drug stimuli pulmonary arterial hypertension, which cannot truly simulate the disease process and disease phenotype of pulmonary arterial hypertension caused by gene mutations
[0005] 2) Most of the existing mouse models are bas

Method used

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  • Preparation method and application of BMPR2 gene mutant rat
  • Preparation method and application of BMPR2 gene mutant rat
  • Preparation method and application of BMPR2 gene mutant rat

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preparation example Construction

[0028] A method for preparing BMPR2 gene mutant rats, the rat BMPR2 gene is mutated, and the mutation site is c.C1471T.

[0029] Further, in a preferred embodiment of the present invention, the following steps are included:

[0030] Synthesize base sequences as the recognition sequences shown in SEQ ID No.1-2, and anneal to form recognition fragments;

[0031] Ligate the recognition fragment with the linearized carrier that has been digested to obtain the Cas9-BMPR2-RNA recombinant vector, transcribe the Cas9-BMPR2-RNA recombinant vector and inject the fertilized egg to obtain the injected fertilized egg;

[0032] The SD female mice were mated with the ligated SD male mice, and the SD female mice with the plug were selected, and the injected fertilized eggs were transplanted into the ampulla of the SD female mice with the plug, to obtain Founder rats;

[0033] The Founder rats were mated with wild-type SD rats to obtain F 1 Generation of rats and screening to obtain F1 gener...

Embodiment 1

[0056] The present embodiment provides a kind of preparation method of BMPR2 gene mutation rat, by the c.C1471T site of mutation rat BMPR2 gene, obtains the rat model that can better simulate pulmonary hypertension; Including synthetic base sequence such as SEQ IDNo. The recognition sequence shown in 1-2; the recognition sequence includes SEQ ID NO.1, namely sgRNA: R-bmpr2-gRNA up35'TAGGgaccaggatgcagaggct;

[0057] SEQ ID NO.2, namely R-bmpr2-gRNA down35'aaacAGCCCTCTGCATCCTGGTC.

[0058] Include the following steps;

[0059] 1.1 Select the c.C1471T (p.R491W) site of the rat BMPR2 gene as the recognition target, design the base sequence of the recognition sequence sgRNA as shown in SEQ ID NO.1-2, and synthesize it as shown in SEQ ID NO.1- The recognition sequence shown in 2;

[0060] 1.2 Annealing and renaturation of the base sequence of the synthesized sgRNA single strand into a recognition fragment; inserting the recognition fragment into the vector backbone treated with BS...

Embodiment 2

[0075] The present embodiment provides a kind of preparation method of BMPR2 gene mutation rat, by the C1471T site of mutation rat BMPR2 gene, obtains the rat model that can better simulate pulmonary arterial hypertension; The recognition sequence shown in 2; the recognition sequence includes SEQ ID NO.1, namely sgRNA: R-bmpr2-gRNA up35'TAGGgaccaggatgcagaggct;

[0076] SEQ ID NO.2, namely R-bmpr2-gRNA down35'aaacAGCCCTCTGCATCCTGGTC.

[0077] Include the following steps;

[0078] 1.1 Select the c.C1471T (p.R491W) site of the rat BMPR2 gene as the recognition target, design the base sequence of the recognition sequence sgRNA as shown in SEQ ID NO.1-2, and synthesize it as shown in SEQ ID NO.1- The recognition sequence shown in 2;

[0079] 1.2 Annealing and renaturation of the base sequence of the synthesized sgRNA single strand into a recognition fragment; inserting the recognition fragment into the vector backbone treated with BSAI digestion and linearization; obtaining the C...

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Abstract

The invention provides a preparation method and an application of a BMPR2 gene mutant rat, belonging to the technical field of biomedicine. The rat BMPR2 gene mutation knocked-in recognition sequenceprovided by the invention is assisted by a gene editing vector, the nucleic acid sequence of BMPR2 gene can be accurately identified by in vitro transcription and then injected into the fertilized egg, and then the gene-edited offspring rats can be obtained by fertilized egg transfer, and then the offspring rats can be obtained by cage mating. The rat model was induced by hypobaric hypoxia and showed severe pulmonary hypertension similar to human disease phenotype, which has high application value.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a preparation method and application of BMPR2 gene mutant rats. Background technique [0002] The pathological mechanism of pulmonary arterial hypertension (PAH) is complex, and the prognosis is extremely poor, with a 5-year survival rate of only 57% (Chest 2012, 142: 448-456). Hereditary pulmonary arterial hypertension (HPAH) refers to pulmonary hypertension caused by specific pathogenic gene mutations. At present, seven PAH pathogenic genes have been found, namely bone morphogenetic protein receptor 2 (Bone Morphogenetic Protein Receptor 2, BMPR2), bone morphogenetic protein receptor 1B (BMPR1B), activin receptor kinase 1 (ACVRL1 / ALK1) , Vascular cell adhesion molecule (Endoglin, ENG), Smad protein 9 (SMAD9), caveolin 1 (CAV1) and potassium ion channel protein 3 (KCNK3). BMPR2 is currently known as the most important causative gene of HPAH, accounting for 20-40% of idiopa...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90A01K67/027A61K49/00
CPCA01K67/0278A01K2217/072A01K2227/105A01K2267/0375A61K49/0008C12N15/8509C12N15/907C12N2800/107C12N2810/10
Inventor 荆志成王晓建李素琪程春燕吕子超刘少飞张思瑾谭元卿周玉平刘倩倩
Owner FUWAI HOSPITAL CHINESE ACAD OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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