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A model and establishment method of CRISPR/Cas9-induced scale-deletion zebrafish

A construction method, zebrafish technology, applied in the field of genetic engineering

Inactive Publication Date: 2018-06-01
CHINA ACAD OF SCI NORTHWEST HIGHLAND BIOLOGY INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

TNF is the functional domain of Eetodysplasin. However, some mutations found in patients show that the collagen region also plays an important role. This region contains 19 G-X-Y repeats, 2 G-X-Y repeats or 4 G-X-Y repeat deletions in the reading frame Although it does not affect the function of the TNF domain, it can also cause EDA syndrome

Method used

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  • A model and establishment method of CRISPR/Cas9-induced scale-deletion zebrafish
  • A model and establishment method of CRISPR/Cas9-induced scale-deletion zebrafish
  • A model and establishment method of CRISPR/Cas9-induced scale-deletion zebrafish

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1 A scale-deleted zebrafish model induced by Crispr / Cas9, which refers to a scale-deleted zebrafish that contains a base insertion at the exon 4 site of the EDA gene.

[0038] The establishment method of the scale-deficient zebrafish model comprises the following steps:

[0039] (1) Using the Ensemble online database, search for the zebrafish EDA gene sequence no: ENSDARG00000074591 and download it, and search for the PAM (protospacer-adjacent motif) sequence in this sequence, namely 5'-GGNNNNNNNNNNNNNNNNNNNNNGG-3', the targeting effect found in the exons of the EDA gene The sequence is TTAGGCAAGAAAGGGCCCCC [TGG], and the mutation detection primer F-eda: ttgttttgcttctcatcagttg and the mutation detection primer R-eda: tttgctctgctgcttcactc are designed and designed.

[0040] (2) Sequencing verification of the wild-type allele of the target sequence:

[0041] Five 24hpf wild-type zebrafish embryos were randomly selected, the genomic DNA template was extracted, and...

Embodiment 2

[0071] Example 2 The application of the scale-deficient zebrafish model to the verification of the function of skin attachment-related genes and the screening of drugs for ectodermal dysplasia.

[0072] PAM sequence

[0073] 5'-GGNNNNNNNNNNNNNNNNNNNGG-3'

[0074] targeting sequence

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Abstract

The present invention relates to a scale-deleted zebrafish model induced by Crispr / Cas9, the model refers to a scale-deleted zebrafish comprising a base insertion at the exon 4 site of the EDA gene. At the same time, the invention also discloses the establishment method and application of the mode. The scale-deficient zebrafish model established by the present invention has great application value in research on the function of genes related to skin appendages, screening and treatment of drugs for ectodermal dysplasia such as human hair thinning.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a model and establishment method of a scale-deleted zebrafish induced by Crispr / Cas9. Background technique [0002] Hair, teeth, nails, sweat glands of humans and mammals, feathers of birds, scales of fish, hypopharyngeal teeth, tentacles, etc. are all skin appendages, and the development of skin appendages is very important to humans, and skin appendages Dysplasia patients have familial heredity, and this phenomenon has also been found in mice and fish. It has been confirmed through genetic studies that the development of skin appendages is mainly regulated by EDA family genes. [0003] Eda (Eetodysplasin) and its receptor Edar is associated with TNF and belongs to the TNF superfamily member. EDA signaling is regulated by Eetodysplasin, EDAR, and EDARADD, forming a TNF ligand in the regulation of vertebrate skin appendages from fish to humans- Receptor-aptamer famil...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/89C12N15/55C12N15/11A01K67/027
Inventor 张存芳赵凯童超刘思嘉汤永涛冯晨光张仁意李国刚
Owner CHINA ACAD OF SCI NORTHWEST HIGHLAND BIOLOGY INST
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