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A method for constructing a genetically modified non-human animal and its application

A technology of non-human animals and construction methods, applied in the fields of biology and biomedicine

Active Publication Date: 2022-05-20
BEIJING CENT FOR DISEASE PREVENTION & CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, there are currently no tools to achieve tissue-specific gene knockout in the brain choroid plexus in non-human animals

Method used

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  • A method for constructing a genetically modified non-human animal and its application
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  • A method for constructing a genetically modified non-human animal and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 Construction of Adgra3-iCreERT2 model rats

[0078] In this example, taking rat ENSRNOT00000036646.6 transcript as an example, iCre-ERT2 was inserted into the Adgra3 gene, so that Adgra3 and iCre-ERT2 were co-expressed, and an Adgra3-iCreERT2 model rat was constructed. It specifically includes inserting the P2A-iCreERT2 element into the region between Exon 24 of the Adgra3 gene and the 3'UTR, wherein the P2A-iCreERT2 element includes GSG-P2A (the amino acid sequence is shown in SEQ ID NO: 21, and the nucleotide sequence shown in SEQ ID NO: 22) and iCre-ERT2 (its amino acid sequence is shown in SEQ ID NO: 17, and its nucleotide sequence is shown in SEQ ID NO: 18). The roadmap for the construction of Adgra3-iCreERT2 model rats is as follows: figure 2 shown. Proceed as follows:

[0079] 1) Amplification and sequencing of the target sequence of the rat genome. Different strains may have different target gene sequences. In order to ensure the efficiency of the...

Embodiment 2

[0106] The Adgra3-iCreERT2 model rats obtained by the method in Example 1 were used to construct PHB1 gene conditional knockout rats. Proceed as follows:

[0107] Intron 3 and intron 5 of rat PHB1 gene were respectively inserted with LoxP sequence to obtain flox rats. Then it was crossed with the Adgra3-iCreERT2 model rat obtained in Example 1 to obtain a rat in which PHB1 was conditionally knocked out.

[0108] Junction PCR and Southern genotype detection were performed on PHB1 conditional knockout rats, and the primers used to construct probes are shown in Table 8.

[0109] Table 8. Primers used to construct probes

[0110]

[0111] Southern results showed that 1EK33-21, 1EK33-28, 1EK33-38, and 1EK33-41 were F1-positive rats, and none of them were randomly inserted (see Figure 4 ).

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Abstract

The invention discloses a method for constructing a genetically modified non-human animal and its application in the field of biomedicine. Specifically, a nucleotide sequence encoding iCre-ERT2 is knocked into the Adgra3 gene, and the core encoding iCre-ERT2 The nucleotide sequence is regulated by an endogenous regulatory element, and a tool for expressing Cre recombinase in the choroid plexus tissue is obtained from a non-human animal. The non-human animal constructed by this application can be used to study the expression of the Adgra3 gene in the brain choroid plexus at different developmental stages and the biological function it may participate in, and as a tool to knock out a specific gene in the non-human animal, and further develop the gene in the The biological functions in the choroid plexus, the possible molecular mechanisms in toxicology and pharmacology, etc. are precisely studied.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a genetically modified non-human animal and its application in the field of biomedicine. Background technique [0002] The choroid plexus (CP) is formed by the differentiation of the ependyma into the ventricle. It 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 pass through the blood-cerebrospinal fluid barrier (BCB, see figure 1 ) Selectively transport some blood-derived substances into the CSF to maintain the homeostasis of the CSF environment, and at the same time prevent the possible damage to the brain tissue caused by some harmful factors in the peripheral circulation. [0003] I...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85A01K67/027C12N15/12C12N15/61C12N15/113
CPCC12N15/8509A01K67/0278C07K14/723C12N9/90C12N15/113C12N2310/20
Inventor 李国君宁钧宇敬海明高珊张楠谭壮生李子南娄云曾晓芃董一文黄蕤赵磊赵可
Owner BEIJING CENT FOR DISEASE PREVENTION & CONTROL
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