Construction method of gys1 and gys2 gene mutants of zebrafish glycogen storage disease

A technology of zebrafish sugar and construction method, which is applied in the field of zebrafish genetics, can solve the problems such as the inability to visually observe the influence of heart development with the naked eye, and the inability to further study the pathogenesis and detailed mechanism of glycogen storage disease.

Active Publication Date: 2022-05-24
SHANGHAI OCEAN UNIV
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Problems solved by technology

[0006] At present, the mutation of human glycogen synthase can lead to human glycogen storage disease, which has a serious impact on human health. There is an MGSKO mouse model of the disease, but the mouse cannot be observed directly with the naked eye due to its own conditions. The effect of Gys1 gene mutation on heart development cannot further study the pathogenesis and detailed mechanism of glycogen storage disease

Method used

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  • Construction method of gys1 and gys2 gene mutants of zebrafish glycogen storage disease
  • Construction method of gys1 and gys2 gene mutants of zebrafish glycogen storage disease
  • Construction method of gys1 and gys2 gene mutants of zebrafish glycogen storage disease

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Experimental program
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Effect test

Embodiment 1

[0041] Zebrafish gys1 and gys2 gene knockout target gRNA design and synthesis, the specific process is as follows:

[0042] a) Sequence analysis of zebrafish knockout genes gys1 and gys2

[0043] According to the annotation of the gys1 and gys2 genes on the Ensembl zebrafish genome website (http: / / asia.ensembl.org / Danio_rerio / Info / Index / ), the gene structures of gys1 and gys2 were obtained, and the similarity of the protein sequences encoded by the two genes was analyzed. sex.

[0044] b) Selection of target sequence

[0045] use T7 in vitro transcription Kit prepares gRNA, selects the target that meets the requirements of T7 in vitro transcription promoter, selects a suitable exon sequence of gys1 and gys2, and uses the zifit website (http: / / zifit.partners.org / ZiFiT / ) to find the target Points, namely: design a target on the 4th exon of the gene gys1, and design a target on the 3rd exon of the gene gys2.

[0046] c) Target gRNA synthesis

[0047] Use T7 promoter to synt...

Embodiment 2

[0074] Wild-type zebrafish parental screening and embryo microinjection, the specific methods are:

[0075] About 20 wild-type zebrafish of the well-grown sexually mature AB strain were selected, and the caudal fins were clipped to extract genomic DNA (DP324, Tiangen Biochemical Technology Co., Ltd.), and PCR products were obtained by amplification with the upstream and downstream primers of the target (Table 1). The PCR product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. If there is no set of peaks at the base site of the sequencing peak map, the amplified region of the sequencing individual is homozygous. Homozygous parents with identical sequences of individual PCR products were selected for microinjection.

[0076] Table 1. Zebrafish gys1 and gys2 gene knockout targets and primer sequence information for detection

[0077]

[0078]The night before microinjection, a pair of male and female zebrafish parents were paired in a mating tank separated ...

Embodiment 3

[0080] F 0 The specific methods for detecting gene knockout efficiency in zebrafish embryos are as follows:

[0081] a) NaOH alkaline lysis method to extract genome

[0082] Take embryos 2 days after injection (5 pieces / tube, 3 tubes in parallel), add 50 μL of 50 mM NaOH to each sample tube, heat at 95°C for 10 min, and shake; repeat heating and shaking; add 5 μL of 1M Tris-HCl, 10,000 r / min , centrifuged for 5 min, and the supernatant is the genome.

[0083] b) PCR reaction

[0084] reaction system:

[0085]

[0086] Reaction program:

[0087]

[0088] c) T7E1 digestion detection

[0089]

[0090] Reaction program:

[0091] 95℃ for 5min, 85℃ for 3s, 25℃ for 5s, 4℃∞

[0092] Add 0.25 μL of T7E1 enzyme (T7E1 enzyme:H 2 Add 0.5 μL of O=1:1 diluent), mix well, and centrifuge, and heat in a water bath at 37°C for 45min.

[0093] 2% agarose gel electrophoresis at 120V, 30min. Observe the electropherogram, and judge whether the knockout is successful according to ...

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Abstract

The invention discloses a method for constructing gys1 and gys2 gene mutants of zebrafish glycogen storage disease, comprising the following steps: selecting the fourth exon sequence of the zebrafish gys1 gene sequence to design the target point for gys1 gene knockout, gys2 gene The third exon sequence of the sequence was used to design the target of gys2 gene knockout, and the amplification primers were designed according to the above target sequence, and the pUC19-scaffold plasmamid was used as the template for PCR amplification, and the gys1 and gys2 genes were obtained by purification and in vitro transcription Knockout the target gRNA, and then introduce the gRNA and Cas9 protein into zebrafish fertilized eggs to obtain homozygous mutants of zebrafish gys1 and gys2 genes with stable inheritance. Zebrafish has a large number of eggs, a short reproductive cycle, and a transparent embryo body, which is helpful for further research on the pathogenesis and detailed mechanism of human glycogen storage disease.

Description

technical field [0001] The invention relates to the technical field of zebrafish gene genetics, in particular to a method for constructing zebrafish glycogen storage disease gys1 and gys2 gene mutants mediated by CRISPR / Cas9 system. Background technique [0002] Glycogen storage disease (GSD) is a group of human congenital disorders of abnormal glucose metabolism that cause a series of different symptoms due to the production disorder of enzymes involved in the synthesis and decomposition of glycogen, mainly involving the liver, muscle and brain. abnormal glycogen metabolism. Glycogen storage diseases have a variety of mutant genes and inheritance patterns. Among them, types I, III, VI, and IX are mainly liver lesions, and types II, V, and VII are mainly muscle tissue damage (Zeng Zhaoqiong, Yi Fan, Xie Xiaobing). . Research progress of glycogen storage disease[J]. Laboratory Medicine and Clinic, 2018, 15(22): 3458-3461.). [0003] Glycogen synthase (GS) is a key enzyme in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/90A01K67/027
CPCA01K67/0276C12N9/1051C12N15/113C12N15/902C12Y204/01011A01K2217/075A01K2207/15A01K2227/40A01K2267/0362
Inventor 任建峰黄姣霍锦倩刘姣祖尧李伟明
Owner SHANGHAI OCEAN UNIV
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