Construction method and application of PHB1 gene knockout non-human animal

A technology for non-human animals and construction methods, applied in the construction and application fields of PHB1 gene knockout non-human animals, capable of solving the problems of undisclosed PHB1 gene knockout non-human animals, undisclosed choroid plexus PHB1 gene knockout non-human animals, etc.

Active Publication Date: 2022-02-01
BEIJING CENT FOR DISEASE PREVENTION & CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] Therefore, the prior art only discloses hepatic PHB1 knockout mice for liver research, but does not disclose choroid plexus PHB1 knockout non-human animals, and has not disclosed the combination of Cre / loxP recombination system and CRISPR / Cas9 system Constructed PHB1 gene knockout non-human animal

Method used

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  • Construction method and application of PHB1 gene knockout non-human animal
  • Construction method and application of PHB1 gene knockout non-human animal
  • Construction method and application of PHB1 gene knockout non-human animal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0147] Example 1 Conditional PHB1 gene knockout Flox rats

[0148] In order to knock out the PHB1 gene, the rat PHB1 gene was mainly knocked out through the Cre / loxP recombination system, and the CRISPR / Cas9 system was used to insert LoxP into the rat PHB1 locus to obtain conditional PHB1 gene knockout Flox rats, and to conditional PHB1 gene Knock out Flox rats and introduce recombinase or recombinase expression sequences to delete the conditional PHB1 gene Knock out the PHB1 gene in Flox animals, so that the PHB1 protein in the choroid plexus tissue does not express or the expressed PHB1 protein has no function, thereby obtaining PHB1 gene knockout Rats, the specific construction method is as follows figure 1 shown.

[0149] Firstly, the CRISPR / Cas system is introduced for gene editing. The target sequence in this system determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 to cut the target gene, but the gene sequence may be different in differen...

Embodiment 2

[0184] Example 2 Conditional PHB1 knockout rats

[0185] In order to obtain rats expressing Cre recombinase in the choroid plexus, specifically, the nucleotide sequence encoding iCre-ERT2 regulated by tamoxifen was knocked into the downstream of the rat Adgra3 gene promoter by using the CRISPR / Cas9 system. The expression of Cre recombinase in the mouse is regulated by the regulatory element of Adgra3 gene, so that the rat specifically expresses Cre recombinase in the choroid plexus tissue.

[0186] The Flox heterozygous rats obtained in Example 1 were crossed with the rats expressing Cre recombinase in the choroid plexus tissue to achieve tissue-specific knockout of the target gene. The specific scheme is as follows: Figure 9 shown. WT means wild-type rats, fl means F1 generation positive heterozygous rats obtained by crossing F0 generation mice with wild-type rats, and Δ means crossing fl rats with tissue-specific Cre or Cre-deleter rats Rats with tissue-specific or system...

Embodiment 3

[0196] Example 3 PHB1 whole gene knockout rat (rat with whole body knockout of PHB1)

[0197] In order to obtain the whole gene knockout rats, the fl heterozygous rats were mated with Cre-deleter rats, the mating scheme is shown in Figure 13 . WT means wild-type rats, fl means F1 generation positive heterozygous rats obtained by crossing F0 generation mice with wild-type rats, and Δ means crossing fl rats with tissue-specific Cre or Cre-deleter rats Rats with tissue-specific or systemic knockout of the gene of interest.

[0198] The first step: mating with Cre-deleter rats to obtain full gene knockout heterozygous rats; the obtained F1 generation heterozygous rats were mated with Cre-deleter rats to remove Exon4-5. The Cre-deleter rats used can be homozygous or heterozygous, such as Figure 14 shown. The genotype detection strategy is shown in Table 11:

[0199] Table 11 The first step genotype detection strategy of whole gene knockout rats

[0200]

[0201] Only het...

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Abstract

The invention relates to a construction method of a PHB1 gene knockout non-human animal and application of the PHB1 gene knockout non-human animal in the fields of toxicology and biological medicine, the non-human animal is obtained by utilizing Cre/LoxP and CRISPR/Cas9 systems, PHB1 protein in venation plexus tissue of the non-human animal is not expressed or the expressed PHB1 protein does not have functions. The PHB1 gene knockout non-human animal can be used as an animal modle for screening medicines for obesity, diabetes, venation plexus related diseases, tumors or inflammations and researching the toxicity of heavy metals or other toxic substances on the venation plexus, and has important application value in research and development of medicines for obesity, diabetes, venation plexus related diseases, tumors or inflammations.

Description

technical field [0001] This application relates to the construction method and application of gene-knockout non-human animals, in particular, to a construction method based on a PHB1 gene brain-knockout non-human animal and its application in the fields of toxicology and biomedicine. Background technique [0002] The choroid plexus (CP) is formed by the differentiation of the ependyma into the ventricle, and is arranged in a network in the ventricle. According to its anatomical location, it can be divided into the lateral ventricle choroid plexus and the third ventricle choroid plexus. And the choroid plexus of the fourth ventricle. The choroid plexus is not only an important source of cerebrospinal fluid (CSF), but also can selectively transport some blood-derived substances into the CSF through the blood-cerebrospinal fluid barrier (BCB) formed by its active epithelium. , in order to maintain the homeostasis of the CSF environment, and at the same time prevent some harmfu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C12N15/55C12N15/113A01K67/027
CPCC12N15/8509C07K14/4703C12N9/22C12N15/113A01K67/0276C12N2800/107C12N2800/30C12N2310/20A01K2217/075A01K2217/15A01K2227/105A01K2267/03A01K2267/0362A01K2267/0368A01K2267/0331
Inventor 李国君敬海明宁钧宇高珊胡红李子南董一文冯颖刘晶晶庞星火刘秀颖黄蕤赵磊赵可
Owner BEIJING CENT FOR DISEASE PREVENTION & CONTROL
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