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Method for preparing atherosclerosis disease canine model

A technology for models and female dogs, which is applied in the field of preparing apolipoprotein E gene knockout disease model dogs using gene knockout technology, can solve the problems of no relevant reports, etc., and achieve the effect of improving utilization efficiency and increasing the number

Active Publication Date: 2017-07-28
北京希诺因生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no relevant report on atherosclerosis gene knockout or transgene modification model dogs

Method used

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  • Method for preparing atherosclerosis disease canine model
  • Method for preparing atherosclerosis disease canine model
  • Method for preparing atherosclerosis disease canine model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction, in vitro transcription and verification of transgene targeting vector

[0054] According to the canine APOE gene sequence information provided by NCBI, the targeting site sequence 5'-CCGGGTGGCAGACTGGCCAGCCC-3' (SEQ ID NO: 4) was selected based on the canine APOE gene Exon3 (see figure 1 ), the sgRNA sequence recognizing this site is 5'-GGGCTGGCCAGTCTGCCACC-3' (SEQ ID NO: 10). When constructing the vector, the backbone carrier T7-gRNA (purchased from Addgene) was digested with BbsI for subsequent experiments; the sgRNA sequence was designed: ataGGGCTGGCCAGTCTGCCACCgt (SEQ ID NO: 5) and the sgRNA complementary sequence: taaaacGGTGGCAGACTGGCCAGCC (SEQ ID NO: 6 ); the sgRNA sequence and the sgRNA complementary sequence are annealed, and then ligated with the digested T7-gRNA plasmid. The T7-sgRNA plasmid was amplified by PCR and the PCR product was recovered, and the PCR product of T7-sgRNA was transcribed in vitro using an in vitro transcription k...

Embodiment 2

[0063] Example 2: APOE gene knockout dog embryo transfer

[0064] A total of 13 beagle bitches in natural estrus were used as fertilized egg donors and embryo transfer recipients. Blood was collected from all bitches to detect the concentration of progesterone in the serum. When the concentration of progesterone reached 4-7ng / mL, it could be determined as the ovulation period. Natural mating was carried out 48 hours after ovulation, and then the fertilized embryos were washed out. 13 bitches were accumulatively fertilized 65 eggs. After collecting the fertilized eggs, use TCM199 medium containing 0.1% hyaluronidase to remove cumulus granulosa cells, then put them into microdrops of HEPES-buffered TCM199 medium (HM, GIBCO11150), and then place them in a micromanipulator equipped with a micromanipulator. instrument on an inverted microscope. Use the microinjection needle to draw the mixed solution containing the mRNA of the sgRNA prepared in Example 1 and the mRNA of Cas9 at a...

Embodiment 3

[0070] Example 3: Detection of gene mutations in APOE knockout dogs

[0071]After puppies were born, ear tissues and tail tissues were collected for identification. After the tissue pieces were shredded in the centrifuge tube, proteinase K was added to a water bath at 56°C for 1-3 hours. Then use a pipette gun to draw 700 μL of Genomic Lysis Buffer, add to the lysis system, mix evenly by inverting up and down, centrifuge at 10,000 g for 1 min. Use a pipette to draw the supernatant to the purification column, 10000g, let stand at room temperature for 1min, and centrifuge for 1min. Replace with a new collection tube, add 200 μL of DNA Pre-Wash Buffer, 10000 g, to the spin column, let stand at room temperature for 1 min, centrifuge for 1 min, and discard the waste. Add 400 μL of g-DNA Wash Buffer, 10000 g, to the spin column, let stand at room temperature for 1 min, centrifuge for 1 min, and discard the waste. Re-centrifuge the purification column and collection tube at 10000g...

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Abstract

The invention relates to a method for preparing an atherosclerosis disease canine model, in particular to a method for preparing an apolipoprotein E (APOE) gene knockout disease canine model according to a gene knockout technology.

Description

technical field [0001] The invention relates to a method for preparing an atherosclerotic disease model dog by gene knockout technology, in particular to a method for preparing an apolipoprotein E gene knockout disease model dog by using gene knockout technology. Background technique [0002] Atherosclerosis (AS) is a multiple disease in the elderly caused by multiple factors such as genetics and environment, and is the main cause of cardiovascular and cerebrovascular diseases such as coronary heart disease, cerebral infarction, and peripheral vascular disease. Coronary atherosclerosis can cause angina pectoris, myocardial infarction, arrhythmia, and even sudden death if the stenosis of the artery reaches more than 75%. [0003] The etiology of AS is complex, and its pathogenesis is related to a variety of pathogenic factors. It mostly occurs in the intima and subintima of large and medium-sized elastic vessels, muscular arterial walls, and is characterized by lipid depositi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/89C12N15/85C12N5/10A01K67/027C12Q1/68C12N15/11
CPCA01K67/0276C07K14/775C12N15/8509C12N15/89C12Q1/6888C12N2810/10C12N2800/107C12Q2600/156A01K2227/10A01K2217/075A01K2267/0375C12N15/873C12N15/113C12N2310/20C12N2015/8527
Inventor 赵建平郑敏冯冲李京李茗瑜陈奔驰
Owner 北京希诺因生物科技有限公司
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