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Construction method of Bestrophin3 vascular smooth muscle specific gene knockout mouse and aortic dissection mouse model

A gene knockout mouse, vascular smooth muscle technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problem of large experimental loss, high mortality rate, and low molding rate of knockout mice and other problems, to achieve the effect of simple modeling and high morbidity

Pending Publication Date: 2022-08-09
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above three methods are widely used, they all have certain shortcomings. The (1) and (2) knockout mice have a low modeling rate, and the disease is mostly in the abdominal aorta, and the pathological manifestations are more similar to the abdominal aorta. The pathological features of aortic dissection are different from those of aortic dissection; the (3) method has a high modeling rate, but there are strict restrictions on the age of the model mice, and 3-week-old mice must be used, which is different from the main clinical practice. The incidence of arterial dissection is inconsistent, and the mortality rate during the modeling period is high, and the experimental loss is large

Method used

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  • Construction method of Bestrophin3 vascular smooth muscle specific gene knockout mouse and aortic dissection mouse model
  • Construction method of Bestrophin3 vascular smooth muscle specific gene knockout mouse and aortic dissection mouse model
  • Construction method of Bestrophin3 vascular smooth muscle specific gene knockout mouse and aortic dissection mouse model

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The construction method of Bestrophin3-fLoxP mouse model specifically includes the following steps:

[0047] (1) Materials: The construction of Bestrophin3-flox mice (C57BL / 6 mice) was entrusted to Saiye Biotechnology Co., Ltd.; the B6.Cg-Tg(Tagln-cre)1Her / J mice used in the experiment were from The Jackson Laboratory; C57BL / 6 mice were obtained from Saiye Biotechnology Co., Ltd.; PCR identification kits were purchased from sigma company in the United States;

[0048] (2) Construction of plasmid vector:

[0049] like figure 1 As shown in the figure, the Cre / LoxP recombination system was used to construct conditional knockout mice. First, a conditional knockout vector of Bestrophin 3 was constructed. Bestrophin 3 gene has 10 exons, ATG is on exon 2, TAA is on exon 10, and Bestrophin 3 is selected. Exon 3 and exon 4 of the gene are used as knockout regions, and deletion of this region should result in loss of function of the mouse Bestrophin 3 gene. Using the C57BL / 6 m...

Embodiment 2

[0059] VerifyBest3 SMKO Mice can spontaneously form AD in the basal state: In order to clarify the role of Bestrophin3 in AD formation, the Cre / LoxP conditional gene targeting technology was used to construct and obtain Bestrophin3 vascular smooth muscle-specific knockout mice (Best3 SMKO ), using Best3 flox+ / + as a control group (Best3 FL / FL ).

[0060] Test procedure: Record Best3 in natural growth state SMKO and Best3 FL / FL The death of mice within 4-72 weeks of age was used to draw the survival curve; the inner diameter of the aorta was detected by ultrasound Doppler of live small animals, and the structural changes of the mouse blood vessels were further clarified by dissecting the blood vessels of the mice by anatomical methods, so as to jointly evaluate the Bestrophin3 Effects of smooth muscle knockout on mouse aortic dissection / aortic aneurysm.

[0061] Test results: as image 3 shown, in the process of rearing the mice, the Best3 SMKO Mice showed slow movement ...

Embodiment 3

[0063] Angiotensin II (Ang II) induces Best3 SMKO Tests for Hypertension and Aortic Dissection in Mice:

[0064] Elevated blood pressure is an important inducement for the development of aortic dissection. In order to simulate the pathological conditions of hypertension at the onset of clinical aortic dissection, 4-week-old mice were subcutaneously implanted with an Ang II sustained-release pump on the back to induce hypertension for 4 weeks. The induction flow diagram is as follows Figure 8 shown, including the following steps:

[0065] (1) To be Best3 FL / FL Mice and Best3 SMKO When the mice were 4 weeks old, two groups of mice from the same litterm were anesthetized by intraperitoneal injection of sodium pentobarbital (100 mg / kg). BW), a micro-osmotic sustained-release pump with Ang II solution (1.75 mg / kg) was implanted subcutaneously on the back, and the release rate of Ang II was 1.44 mg / kg / d. The systolic blood pressure (SBP) and diastolic blood pressure (DBP) of t...

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Abstract

The invention discloses a construction method of Bestrophi n3 vascular smooth muscle specific gene knockout mouse and aortic dissection mouse models, and relates to the field of mouse model establishment. The aortic dissection mouse model is constructed by the following steps: adopting a Bestphi n3 vascular smooth muscle specific gene knockout mouse, called Best3SMKO mouse for short, and feeding the Best3SMKO mouse for 24 weeks to spontaneously form aortic dissection or aortic aneurysm. According to the application, aortic dissection can be induced by deletion of Bestrophi n3 in vascular smooth muscle cells, the constructed Bestrophi n3 smooth muscle specific knockout mouse is an aortic dissection / aneurysm mouse model which is high in morbidity, similar to the human aortic dissection morbidity in age and high in quality, the AD morbidity of the Best3SMKO mouse reaches 63%, the morbidity is high, and modeling is simple.

Description

technical field [0001] The invention relates to the field of mouse model establishment, in particular to a method for constructing a Bestrophin3 vascular smooth muscle-specific gene knockout mouse and aortic dissection mouse model. Background technique [0002] Aortic dissection (AD) is a common acute vascular disease, and the incidence is increasing year by year, accompanied by the younger age of cardiovascular diseases such as hypertension. The development of aortic dissection is very dangerous. Once the disease occurs, if it is not treated in time, the blood vessel will rupture, which will cause cardiac tamponade, acute tracheal hemorrhage, and shock death. Once the blood vessels of aortic dissection patients are ruptured, artificial blood vessels can only be replaced by surgery. The replacement faces huge surgical risks, and the blood vessels will further degenerate after the operation, and face the risk of secondary surgery and rupture to prevent aortic rupture. At pre...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/12A01K67/027A61K49/00
CPCC07K14/47C12N15/8509A01K67/0276A61K49/0008A01K2217/075A01K2227/105A01K2267/03Y02A50/30
Inventor 周家国张亭亭吕晓飞梁思佳雷青青
Owner SUN YAT SEN UNIV
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