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Construction method of Cyp1a1 gene knockout mouse model and application of Cyp1a1 gene knockout mouse model in sepsis

A gene knockout mouse and construction method technology, applied in the field of biomedicine, can solve problems such as ambiguity, and achieve the effects of improved viability, high knockout efficiency, and enhanced bacterial removal ability

Active Publication Date: 2020-12-04
中国人民解放军陆军特色医学中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, what role and function CYP1A1 plays in the metabolic disorder of sepsis is still unclear, and there is no evidence that CYP1A1 participates in the regulation of the "arginine-agmatine-polyamine / Research Report on Metabolic Axis of GABA

Method used

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  • Construction method of Cyp1a1 gene knockout mouse model and application of Cyp1a1 gene knockout mouse model in sepsis
  • Construction method of Cyp1a1 gene knockout mouse model and application of Cyp1a1 gene knockout mouse model in sepsis
  • Construction method of Cyp1a1 gene knockout mouse model and application of Cyp1a1 gene knockout mouse model in sepsis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction and Identification of Cyp1a1-KO Mouse Model

[0029] 1. Experimental animals

[0030] The mouse strain used in this example is: C57BL / 6J, and the surrogate mother mouse strain is C57BL / 6J.

[0031] 2. Experimental method

[0032] 2.1 Knockout mouse construction method

[0033] 2.1.1 Construction of sgRNA plasmid expressing mouse Cyp1a1 gene exon2-7

[0034] The gene sequence of Cyp1a1 gene exon2-7 and the designed sgRNA information are as follows:

[0035] Exon2-7 sequence:

[0036]

[0037]

[0038]

[0039]

[0040] The underlined part in the above gene sequence (SEQ ID No: 1) represents the exon region, and the ununderlined part represents the intron region.

[0041] The sgRNA sequence is as follows:

[0042] Cyp1a1-Sg1: CACCTCTCTTAATTGTG (SEQ ID No: 2)

[0043] Cyp1a1-Sg2: CTCTAGAAGTGGGCTACTT (SEQ ID No: 3)

[0044] Cyp1a1-Sg3: TCATTCCTTTACTCTAGACC (SEQ ID No: 4)

[0045] Cyp1a1-Sg4: GCTGTTTAGCCTCAGC (SEQ ID No: 5)

[0046]...

Embodiment 2

[0071] Example 2 Application of Cyp1a1-KO mouse model

[0072] In order to better demonstrate the application of the Cyp1a1-KO mouse model of the present invention, the experimental methods given below are all better or optimal implementations of mouse model establishment.

[0073] 1. Analysis of bacterial clearance ability of Cyp1a1 knockout mice

[0074] 1.1 Experimental method

[0075] The ability of Cyp1a1 knockout mice to clear Escherichia coli was analyzed by counting bacteria in peripheral blood.

[0076] 1.1.1 Activate Escherichia coli in sufficient quantity overnight, wash twice with saline and resuspend, then calculate the concentration of Escherichia coli by colorimetry.

[0077] 1.1.2 Random number table method to select 12 6-8 weeks old, similar weight, male Cyp1a1 + / + and Cyp1a1 - / - mice, each mouse was injected 5x 10 via the tail vein 7 CFU E. coli.

[0078] 1.1.3 After 6 hours, the mice were sacrificed, the peripheral blood was taken, diluted 100 times, e...

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Abstract

The present invention discloses a construction method of a Cyp1a1 gene knockout mouse model and an application of the Cyp1a1 gene knockout mouse model in sepsis. The construction method comprises thefollowing steps: determining target sites of a mouse gene to be knocked out and designing sgRNAs, wherein the sequences of the sgRNAs are shown as SEQ ID No:2-5; co-injecting or co-electrically transferring the active sgRNAs and Cas9 mRNA or Cas9 protein into fertilized egg cells of mice and transplanting the fertilized eggs into a receptor mother mouse for pregnancy to obtain F0-generation gene knockout mice; and hybridizing the F0-generation mice with wild-type mice to obtain F1-generation Cyp1a1 gene knockout mice. The Cyp1a1 gene knockout mouse model is successfully prepared and applied toa pathology process research of sepsis for the first time, it is accidentally found that a bacteria-removing capacity of the Cyp1a1 gene knockout mice is remarkably enhanced, viability is improved when the Cyp1a1 gene knockout mice are attacked by endotoxemia and gram-negative bacteria, and an anti-infection capacity is enhanced. The Cyp1a1 gene knockout mice are applied to metabolic regulation and control researches of the sepsis for the first time, and the reliable animal model is provided for related metabolic mechanism researches in the field of critical diseases.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for constructing a Cyp1a1 gene knockout mouse model, and the application of the Cyp1a1 gene knockout mouse in sepsis. Background technique [0002] Sepsis 3.0 defines sepsis in humans as life-threatening organ dysfunction caused by a dysregulated body response to infection. With the gradual understanding of the nature of sepsis, it has been realized that the pathogenesis of sepsis is closely related to infection, inflammation, coagulation, neuro-endocrine-immune response, etc., and involves abnormal changes in multiple organs and systems throughout the body. [0003] Disruption of metabolic pathways, host hypoxic response, and overdrive of the immune system are pathological hallmarks of sepsis at the molecular level. While the mechanisms of the inflammatory response in the pathogenesis of sepsis are well understood, those leading to metabolic dysregulation and multi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/89A61D19/04A01K67/027
CPCC12N15/113C12N15/89A61D19/04A01K67/0276A01K2207/15A01K2217/075A01K2227/105A01K2267/0337
Inventor 马晓媛王芳杰唐婉琪李卫杨雪梁华平
Owner 中国人民解放军陆军特色医学中心
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