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Construction method and application of Tecrl knockout mouse model

A technology of mouse model and construction method, applied to other methods of inserting foreign genetic materials, chemical instruments and methods, biochemical equipment and methods, etc.

Pending Publication Date: 2022-05-13
SHANGHAI CHILDRENS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is still no mouse model of CPVT with Tecrl mutation

Method used

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  • Construction method and application of Tecrl knockout mouse model
  • Construction method and application of Tecrl knockout mouse model
  • Construction method and application of Tecrl knockout mouse model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] This embodiment provides a method for constructing a Tecrl gene knockout mouse, comprising the following steps:

[0041] 1.1 Design and identification of sgRNA

[0042] The key to gene knockout lies in the selection of the target, acting on the correct target can cause the loss of gene function through sequence mutation. In this example, after determining the target of gene knockout, the CRISPR Design software (http: / / crispr.mit.edu / ) was used to design 8 pairs of sgRNAs (named sgRNA1~sgRNA16) against the Tecrl gene, and the sgRNAs were designed according to the Corresponding primers were synthesized by sequence and ligated into the pCS vector by annealing and polymerization. Then, the activity of sgRNA was detected by using the CRISPR / Cas9 activity detection method independently developed by BioStudio-UCATM, and the results were as follows Figure 8 As shown, the screening identified two sgRNAs with higher activity. Specifically, sgRNA8 and sgRNA14, the sequences of...

Embodiment 2

[0058] The F3 generation Tecrl that embodiment 1 obtains is knocked out (Tecrl - / - ) After the mouse model was fed normally for 6-8 weeks, the mice were anesthetized under 3.5ml / min isoflurane, and the Vevo2100 small animal ultrasound imaging system was used to perform ultrasonic examination on the 6-8 week Tecrl knockout mice. The heart obtained M-mode echocardiographic images and related indicators of cardiac function. The result is as figure 2 and image 3 As shown, compared with wild-type C57BL / 6 mice (WT), Tecrl knockout mice (KO) had significantly lower cardiac ejection fraction and short-axis ratio. During diastole, compared to wild-type mice (WT), Tecrl - / - The isovolumic relaxation time of mice (KO) was significantly prolonged, and the end-diastolic diameter of the left ventricle was significantly increased. This result shows that Tecrl knockout mice exhibited decreased left heart function with impaired systolic and diastolic function at 6-8 weeks, and this mouse...

Embodiment 3

[0060] The F3 generation Tecrl that embodiment 1 obtains is knocked out (Tecrl - / - ) After the mouse model was raised normally for 6-8 weeks, epinephrine (ISO) and caffeine were injected simultaneously by intraperitoneal injection. Choose between 3mg / kg, can make Tecl - / - Mice showed obvious bidirectional ventricular tachycardia). The electrocardiogram data of the mice were collected before and after the injection to compare the ECG changes of the mice before and after the injection of epinephrine and caffeine. The specific method for collecting the ECG data of the mice is as follows: anesthetize the mice under 3.5ml / min isoflurane, fix them on the ECG recording board in the supine position, fix the limbs with adhesive tape, approach the surface leads of the ECG, and use the electrocardiogram The software collects the ECG data of mice. Wild-type C57BL / 6 mice were used as controls.

[0061] The result is as Figure 4-Figure 7 As shown, before injecting caffeine and epineph...

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Abstract

The invention provides a construction method and application of a Tecrl knockout mouse model, and the construction method of the Tecrl knockout mouse model comprises the following steps: A, designing sgRNA aiming at a Tecrl gene according to a gene knockout target; and B, injecting Cas9mRNA and sgRNA into a fertilized egg of an experimental mouse at the same time to obtain an F0-generation mouse of which the Tecrl gene is successfully knocked out. According to the invention, a Tecrl knockout mouse model is constructed for the first time, and it is found that the congenital mouse gene defect causes damage to the systolic and diastolic functions of the mouse in the early stage, so that a model is provided for researching the left heart function. The Tecrl knockout mouse has polymorphic and bidirectional ventricular tachycardia under induction of epinephrine and caffeine, clinical manifestations of CPVT patients in a movement or pressure state are simulated clinically, and a powerful tool is provided for studying the pathophysiological mechanism of catecholamine sensitive ventricular tachycardia.

Description

technical field [0001] The invention relates to the technical field of mouse model construction, in particular to a method for constructing a Tecrl knockout mouse model and its application. Background technique [0002] Sudden cardiac death caused by arrhythmia is the leading cause of death in patients without cardiac organic changes. Catecholamine-sensitive ventricular tachycardia (CPVT) is a hereditary ion channelopathy, which mainly occurs in children aged 7-12, with an incidence rate of about 1 / 10000. Its main clinical feature is polymorphic or bidirectional ventricular tachycardia induced by exercise or emotional agitation. The first symptom of some patients with catecholamine-sensitive VT is syncope or sudden death. However, the specific pathogenesis is still unclear. [0003] The diagnosis of catecholamine-sensitive ventricular tachycardia mainly depends on the characteristic electrocardiogram and genetic testing. For patients with typical CPVT, ECG exercise stres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/89A01K67/027C12N15/113A61K49/00
CPCC12N15/89A01K67/0276C07K14/47C12N15/113A61K49/0008A01K2227/105A01K2267/0375C12N2310/20
Inventor 侯翠兰肖婷婷谢利剑林舒嘉郑钧敏陈顺邱庆竹
Owner SHANGHAI CHILDRENS HOSPITAL
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