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

[0045] Example 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 mouse genome as a template to amplify the upstream homology arm, downstream homology arm and flox region, insert loxP into the Bestrophin3 gene intron2, insert the FRT-Neo expression cassette-FRT-loxP into the intron4, and connect to the backbone , the schematic diagram of the LoxP insertion site is shown in figure 2 As shown, a targeting vector (with Neo marker gene) is constituted, and the nucleotide sequence of the targeting vector is shown in SEQ ID NO: 1.

[0050] (2) The above-mentioned targeting vector was linearized and transfected into embryonic stem cells of C57BL/6 mice, and the embryonic stem cells with successful homologous recombination were identified and screened by PCR. The primer sequences used in PCR were shown in Table 1, and the positive embryonic stem cells were subsequently Microinjection into blastocysts and transplantation into pseudopregnant mice.

[0051] (3) F1 generation mice were obtained by passage, and the obtained Bestrophin3-flox heterozygous mice were identified by PCR. The primer sequences used for PCR are shown in Table 1.

[0052] (4) Breeding and screening of Bestrophin3 vascular smooth muscle-specific knockout mice:

[0053] The above-mentioned Bestrophin3-flox heterozygous mice were self-bred and screened by PCR to obtain Bestrophin3-flox homozygous mice, which were then cross-bred with vascular smooth muscle cre tool mice, namely B6.Cg-Tg(Tagln-cre)1Her/J small mice. Mouse, referred to as Tagln-Cre mouse. The obtained F1 hybrid mice were weaned at 3 weeks and their genotypes were identified by the rat tail method. The Bestrophin3 chimeric flox heterozygous and Tagln-Cre positive mice were crossed with Bestrophin3-flox homozygous mice again.

[0054] (5) Carry out PCR identification to produce the mice obtained, the primer sequences used in PCR are shown in Table 1, and the screening can obtain the control group (Bestrophin3-flox homozygous and Tagln-Cre negative, namely Best3 FL/FL mice) and experimental group (Bestrophin3-flox homozygous and Tagln-Cre positive, namely Best3 SMKO mice), which can be modeled for aortic dissection at 4 weeks of age.

[0055] Table 1 - primer sequences used in PCR identification in Example 1

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[0057]

[0058] 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 and unexpected death. The death of mice of different ages was recorded and counted, and Best3 knockout could shorten the survival time of mice and significantly increase the mortality rate. Best3 that will die unexpectedly SMKO Mice were immediately dissected, e.g. Figure 4 As shown, severe hemorrhage in the thoracic cavity was found, and the aortic dissection was obvious after dissection of the aorta, and abdominal aortic rupture occurred. like Figure 5 As shown, the abdominal aorta (AbAo) was monitored by intravital ultrasound in two groups of 4-week, 8-week, 12-week, 16-week, 20-week, 24-week, 48-week, and 72-week-old mice, respectively, to monitor the aortic structure. and ascending aorta (Arch) and measure vessel diameter; such as Figure 6-7 shown, and anatomical observations were made. The results showed that the aorta of mice with Bestrophin3 knockout formed aortic clipping in the early stage, that is, the early signs of aortic dissection, and wall hematoma occurred. Further damage results in the formation of a dissecting aneurysm.

[0062] 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 the mice were tracked and detected with a non-invasive blood pressure meter for 7 days, 14 days, 21 days, and 28 days, and the survival curves of the mice were calculated as follows: Figure 9 As shown, the survival rate after 28 days was 78%.

[0066] (2) Ultrasound detection of mice was collected using VEVO 3100 ultrasound equipment (Visual Sonics, Toronto, Canada). First, the mice to be tested were placed in an anesthesia box with 2% isoflurane until coma, and then fixed on a carrier plate with 1% isoflurane. Continuous anesthesia with isoflurane, select the appropriate body position to collect images of the ascending aorta, aortic arch, thoracic aorta and abdominal aorta, observe the structure of the entire aorta and whether an aortic aneurysm is formed, and measure its maximum inner diameter. The image results are as follows Figure 10 shown. The samples were dissected at the same time, and the results were as follows: Figure 11 shown.

[0067] Test results: as Figure 10 As shown, on the 28th day after induction of hypertension, the mouse aorta was scanned by ultrasound to observe its wall structure, and Best3 SMKO The mouse aorta forms arterial dilation in the ascending aorta, abdominal aorta, etc., and produces dissecting aneurysm, Best3 SMKO The diameter of the aorta of the mice increased significantly. Further anatomical observations, such as Figure 11 As shown, the anatomical map can be seen Best3 SMKO In the abdominal aorta, there is a high incidence of aortic dissection and the formation of tumor-like enlargement. Statistics such as Figure 12 shown, Best3 SMKO The incidence of AD in mice was 63%, much higher than Best3 FL/FL Group.