Statla gene deletion type zebra fish

A gene deletion and zebrafish technology, applied in genetic engineering, biochemical equipment and methods, animal husbandry, etc., can solve the problems of low efficiency of targeting technology and high off-target rate

Inactive Publication Date: 2016-05-25
HUNAN NORMAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

Traditional gene targeting technology is based on embryonic stem cells (ESC) and homologous recombination technology, so the efficiency of targeting technology is extremely low
At the beginning of 2013, a new artificial endonuclease clusteredregularlyinterspacedshortpalindromicrepeats (CRISPR) / CRISPR-associated (Cas) 9, which can more efficiently and accurately silence specific genes in the genome of organisms, is simple to make, low in cost, and can be Simultaneously cut multiple sites on the target gene to silence any number of single genes, but at the same time, this technology has certain defects, and its off-target rate is relatively high

Method used

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  • Statla gene deletion type zebra fish
  • Statla gene deletion type zebra fish
  • Statla gene deletion type zebra fish

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Embodiment Construction

[0090] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0091] Such as Figure 1-3 As shown, a stat1a gene deletion type zebrafish of the present invention consists of the following steps:

[0092] A. CRISPR / Cas9 gene knockout target site design

[0093] Query the genomic DNA sequence and functional domain of the zebrafish stat1a gene on National Center for Biotechnology Information (NCBI), and design a pair of stat1a on the website TheZiFiTTargeter (http: / / zifit.partners.org / ZiFiT_Cas9) according to the principle of CRISPR / Cas knockout The target site of the gene. The selection of targets must follow this standard: 5'-GG-(N)18-NGG-3'. The GG dinucleotide at the 5' end is part of the T7 promoter, and this restriction is not required when designing the target site, but it must be ensured that the 3' end of the target site is NGG. The selection of the target must ensure that the insertion or del...

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Abstract

The invention provides statla gene deletion type zebra fish. After an experiment design region is knocked out, the sequence is represented as SEQ ID No.1; an experiment comprises the following steps: designing a CRISPR / Cas9 gene knockout target point: constructing a gRNA expression vector and synthesizing gRNA in vitro; carrying out micro-injection of zebra fish embryos; detecting the effectiveness of the target point by a T7E1 method and Sanger sequencing; two months after injection, carrying out tail shearing and identification and carrying out identification steps above; carrying out TA cloning of a target sequence; carrying out Sanger sequencing of plasmids; obtaining an F1 generation of descendible zebra fish mutants; obtaining F2 generation homozygotes of the zebra fish mutants; and carrying out F3 generation homozygous heredity of the gene deletion type zebra fish according to the method above to obtain a new zebra fish line.

Description

technical field [0001] The invention belongs to the technical field of gene knockout, and relates to a stat1a gene-deleted zebrafish. Background technique [0002] STAT1 (signaltransducerandactivatoroftranscription1) gene is located in human 2q32.3, which encodes a transcription factor of 750 amino acids. It is generally believed that this gene mediates the interferon signaling pathway, can directly regulate the transcription of target genes, and participate in processes such as cell proliferation and differentiation. Through gene differential expression profile analysis and genome association analysis, it was found that STAT1 gene was closely related to osteoporosis. [0003] The genes and signaling pathways in the skeletal development process between zebrafish and humans are highly homologous, and the STAT1 gene is relatively conservative in evolution. The two different splice forms STAT1-alpha and STAT1-beta of the human STAT1 gene correspond to the two Different genes ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C12N15/85C12Q1/68
CPCA01K67/0276A01K2217/075A01K2227/40A01K2267/02C07K14/461C12N15/8509C12Q1/6888C12Q2600/156
Inventor 陈湘定苏幸熊玖玲邓云邓红文
Owner HUNAN NORMAL UNIVERSITY
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