Construction method of animal model with liver HO-1 gene conditionally knock out by utilizing CRISPR-Cas9

A HO-1, animal model technology, applied in the field of genetic engineering

Active Publication Date: 2020-05-29
THE THIRD HOSPITAL OF HEBEI MEDICAL UNIV
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  • Description
  • Claims
  • Application Information

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  • Construction method of animal model with liver HO-1 gene conditionally knock out by utilizing CRISPR-Cas9
  • Construction method of animal model with liver HO-1 gene conditionally knock out by utilizing CRISPR-Cas9
  • Construction method of animal model with liver HO-1 gene conditionally knock out by utilizing CRISPR-Cas9

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Embodiment 1

[0023] The liver HO-1 gene knockout animal model was commissioned by the Nanjing Institute of Biomedicine, Nanjing University to construct:

[0024] 1. Design, construction and purification of sgRNA of mouse Hmox1(HO-1) gene

[0025] Design Hmox1 conditional knockout mouse strategies such as figure 1 shown. According to the strategy, determine the flox region as exon2 of the Hmox1 gene, use the CRISPR Design tool of MIT (http: / / crispr.mit.edu / ), design a pair of 20bp DNA targeting Hmox1 gene according to the level of Score The oligonucleotide chain sequence is used to prepare sgRNA, and the designed sequence is synthesized into a PAGE product. According to the sgRNA sequence, a Donor vector carrying the homologous region of the target site and the loxp site was designed, and primers were designed in the target region for subsequent gene identification of mice. Order the corresponding Oligo sgRNA sequence as follows:

[0026] Table 1 Oligo sgRNA sequences

[0027] ...

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Abstract

The invention provides a construction method of an animal model with a liver HO-1 gene conditionally knock out by utilizing CRISPR-Cas9, belonging to the technical field of gene engineering. The method comprises the following steps: 1) introducing an sgRNA sequence, a Cas9 gene sequence and a donor gene sequence into fertilized eggs of a mouse to obtain an F0-generation mouse containing a targeting sequence; 2) hybridizing the F0-generation mouse containing the targeting sequence obtained in the step 1) with a wild-type mouse to obtain a heterozygous mouse containing the targeting sequence; and 3) hybridizing the heterozygous mouse containing the targeting sequence obtained in the step 2) with an Alb-Cre positive mouse to obtain the liver HO-1 gene knockout animal model.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing an animal model for conditionally knocking out the HO-1 gene by using CRISPR-Cas9. Background technique [0002] Heme oxygenase-1 (heme oxygenase-1, HO-1, Hmox1) is the initial enzyme and rate-limiting enzyme of heme metabolism. Its main function is to catalyze the production of bilirubin, carbon monoxide and free iron from heme, and reduce hemoglobin Direct damage to the cell membrane by toxins and cell damage caused by oxygen free radicals produced. Current studies have shown that the increased expression of HO-1 is a protective mechanism for the body to resist oxidative stress and lipid peroxidation, and it can protect liver cells and resist various liver injuries such as oxygen poisoning, lipopolysaccharide, and ischemia-reperfusion. Inflammatory and anti-fibrotic effects. The stress ability of HO-1-deficient mice is reduced...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/53A01K67/027
CPCC12N15/907C12N9/0071A01K67/0276C12Y114/14A01K2217/075A01K2227/105A01K2267/03
Inventor 南月敏杜静华王荣琦李冬冬苑喜微
Owner THE THIRD HOSPITAL OF HEBEI MEDICAL UNIV
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