GRNa gene knockout zebrafish mutant and preparation method thereof

A gene knockout and zebrafish technology, applied in the field of gene editing, can solve problems such as expensive, complicated operation, and long cycle, and achieve good commercial value

Pending Publication Date: 2020-03-24
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the past, gene knockout methods commonly used gene-trap, ZFN, and TALEN, etc., but these techniques are complicated to operate, take a long time, and are expensive

Method used

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  • GRNa gene knockout zebrafish mutant and preparation method thereof
  • GRNa gene knockout zebrafish mutant and preparation method thereof
  • GRNa gene knockout zebrafish mutant and preparation method thereof

Examples

Experimental program
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preparation example Construction

[0036] This embodiment provides a kind of preparation method of GRNa gene knockout zebrafish strain, comprising the following steps:

[0037] (1) Screening sgRNA targeting GRNa gene

[0038] Find the genome sequence of the zebrafish GRNa gene from NCBI, and use the sgRNA design software (https: / / crispr.cos.uni-heidelberg.de / ) to select five sgRNA binding sites in the exon region of the coding protein ( figure 1 ) and design corresponding sgRNA primers, and anneal the sgRNA primers and connect them to the sgRNA expression vector;

[0039] The five sgRNA sites are respectively:

[0040] sgRNA1: TTTGTCCATTGCTGTCCTA;

[0041] sgRNA2: TTATGTACCGAAGAGCCCT;

[0042] sgRNA3: CTACTACTCCCAATGTGAT;

[0043] sgRNA4: AAATGTGACGTAGCTGCG;

[0044] sgRNA5: GTGCCCGTCCGTCCAATC.

[0045] (2) Preparation of sgRNA and Cas9 mRNA of zebrafish GRNa gene

[0046] The sgRNA and Cas9 mRNA of the zebrafish GRNa gene were obtained by in vitro transcription using an in vitro transcription kit (L...

Embodiment

[0059] (1) The zfGRNa sgRNA linker was added with enzyme-cut cohesive terminal bases to facilitate ligation into the expression vector, and primers were synthesized by Huada Gene Company. The primer sequences are shown in the following table:

[0060]

[0061]

[0062] Each pair of sgRNA fragments was annealed at room temperature into the pT7 / sgRNA backbone vector linearized with BbsI to obtain the pT7 / zfGRNa sgRNA expression vector.

[0063] (2) Preparation of sgRNA and Cas9 mRNA of zebrafish GRNa gene

[0064] The purchased pT7-Pt3ts-nCas9n plasmid (commercially available addgene, #64237) was linearized with SpeI enzyme, and the reaction system was as follows:

[0065]

[0066] Reaction conditions: 37°C, 2 hours.

[0067] Take 2 μL of the digested product and add 2 μL 10×DNA Loading Dye and 16 μL DW, add the sample and DL15000 DNAMarker into the well of the nucleic acid gel, and perform electrophoresis under the condition of 160V. When observed in the nucleic acid ...

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Abstract

The invention relates to a preparation method of a GRNa gene knockout zebrafish strain, which comprises the following steps: screening sgRNA of a target GRNa gene, constructing a eukaryotic expressionvector carrying Cas9 and sgRNA expression elements, screening a positive zebrafish founder, and screening first filial generation and second filial generation. The expression condition of the GRNa gene is identified and the obtained GRNa gene knockout zebrafish strain is proved to normally mate and lay eggs and can be stably inherited to filial generations after being mutually intercrossed for more than three generations of the GRNa gene knockout zebrafish strain. And genome, mRNA and protein levels prove that the GRNa gene is hardly expressed. The invention can be used as a tool for zfGRNa gene research. Related experiments such as HE staining and immunohistochemistry prove that the GRNa gene knockout zebrafish strain shows an obvious pathological phenotype and can be used for researching mechanisms of GRNa in diseases such as nervous system diseases and metabolic diseases and screening related drugs; the invention can also be applied to scientific research and economic culture of zebrafish, and has good commercial value.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and specifically relates to a GRNa gene knockout zebrafish mutant and a preparation method thereof, which can be used as a tool for GRNa gene function research and related drug screening. Background technique [0002] Progranulin (PGRN) is a multifunctional growth factor. The heterozygous deletion of this gene is associated with frontotemporal lobar degeneration (FTLD), while the homozygous deletion is considered to be the cause of NCL (neuronal ceroid lipofuscinosis) pathogenic factors. The gene has been extensively studied in human disease and mouse models, but little has been studied in zebrafish. Zebrafish has biological characteristics such as fast growth and development, short reproductive cycle, large number of offspring, in vitro fertilization, and transparent embryos, which are very suitable for gene editing. [0003] There are four homologs of this gene in zebrafish, GRNa, GRNb, ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/89C12N15/90A01K67/027A01K67/02
CPCA01K67/02A01K67/0276A01K2207/15A01K2217/075A01K2227/40A01K2267/0318C12N15/113C12N15/89C12N15/902C12N2310/20
Inventor 夏海滨朱久玲
Owner SHAANXI NORMAL UNIV
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