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Construction method of mouse with Foxp3 gene knocked out under conditions

A construction method, foxp3 technology, applied in the field of construction of conditional knockout Foxp3 gene mice

Inactive Publication Date: 2020-10-09
THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the existing models, there is no special regulation on the labeling of exons, and some label the exon where ATG is located as exon1, which is somewhat different from the representation method in the current database

Method used

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  • Construction method of mouse with Foxp3 gene knocked out under conditions
  • Construction method of mouse with Foxp3 gene knocked out under conditions
  • Construction method of mouse with Foxp3 gene knocked out under conditions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] 1. Targeting strategy:

[0076] flox exon2-8, 3.5kb, refer to the existing model [Fontenot JD, et al., Foxp3 programs the development and function of CD4+CD25+regulatory T cells.Nat Immunol.2003 Apr;4(4):330-6)] , theoretically no protein will be produced after knockout, but the promoter region is not knocked out, and it is possible to find ATG downstream to continue translation. In the literature, there is no expression by Western blot detection; the design scheme uses the Cre-loxP system, and the conditional knockout mouse is constructed during the construction process , loxP is usually inserted into the downstream intron of the ATG-containing exon, and the removal of the spanning exon will cause the shift of the protein reading frame. After searching the FOXP3 gene structure, it was found that

[0077] exon 2(ENSMUSE00000704001),

[0078] exon 3 (ENSMUSE00000704000),

[0079] exon 4 (ENSMUSE00000703999),

[0080] exon 5 (ENSMUSE00000245581),

[0081] exon 6 (ENS...

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Abstract

The invention provides a construction method of a mouse with Foxp3 gene knocked out under conditions. The invention belongs to the technical field of gene engineering. The method comprises the following steps of: respectively carrying out in vitro transcription on EGE-ZXC-007-sgRNA1 and EGE-ZXC-007-sgRNA9 to obtain RNA1 and RNA9, introducing RNA1 and RNA9 and a targeting vector into fertilized eggs of mice to obtain an F0-generation mouse, and carrying out flox genotype detection to obtain an F0-generation positive mouse; hybridizing the F0-generation positive mouse with a wild mouse to obtainan F1-generation mouse, and detecting the flox genotype of the F1-generation mouse to obtain an F1-generation positive mouse; hybridizing the F1-generation positive mouse with a tissue-specific Cre mouse to obtain a floxed heterozygote mouse; and hybridizing the floxed heterozygote mouse with a Cre-deeter mouse to obtain the mouse with the Foxp3 gene knocked out. By adopting the method provided by the invention, the Foxp3 gene in the mouse can be knocked out in a specific tissue.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing conditionally knocked out Foxp3 gene mice. Background technique [0002] Foxp3 is an X-chromosome-encoded transcription factor, belonging to the transcriptional regulator of forkhead / winged-helixfamily, which plays an important role in the development, differentiation and function maintenance of Treg cells, and is currently one of the most ideal Treg markers. Alexander et al. confirmed in 2010 that the conserved non-coding DNA sequences CNS1, CNS2 and CNS3 in the Foxp3 gene control the development and differentiation of mouse Tregcells. Among them, CNS2 is rich in CpG, which is specifically demethylated in mature Treg, and its methylation status is related to the expression and stability of Foxp3. CNS2 knockout mice spontaneously develop lymphoproliferative diseases with multi-organ inflammatory lesions . The gene knockout mice ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/12A01K67/027
CPCA01K67/0276A01K2217/077A01K2227/105C07K14/47C12N15/85C12N15/907
Inventor 任贺高超郎鸣晓
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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