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Transgenic zebrafish model with G6PD (gluocose-6-phosphate dehydrogenase deficiency) 118-144 site defects

A transgenic, zebrafish technology applied in the field of genetic engineering

Inactive Publication Date: 2016-09-07
GUIZHOU MEDICAL UNIV
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Problems solved by technology

[0004] At present, there is no ideal animal experimental model that can be well realized g6pd Research on the relationship between gene structure change and function

Method used

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  • Transgenic zebrafish model with G6PD (gluocose-6-phosphate dehydrogenase deficiency) 118-144 site defects
  • Transgenic zebrafish model with G6PD (gluocose-6-phosphate dehydrogenase deficiency) 118-144 site defects
  • Transgenic zebrafish model with G6PD (gluocose-6-phosphate dehydrogenase deficiency) 118-144 site defects

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Embodiment 1

[0028] The invention provides a method for obtaining transgenic zebrafish by microinjecting plasmids into embryos, and constructs gata1- g6pd M118-144 -egfp-PBSK-Isce I Plasmid, the newly constructed plasmid and Isce I enzyme were injected into zebrafish 1-cell stage embryos together by microinjection, reaching gata1 promoter driver mutation g6pd gene and egfp Effects of gene specificity on erythroid cell expression.

[0029] Wherein, the method for preparing the G6PD transgenic zebrafish model comprises the following steps:

[0030] (1) Prepare mutants by overlapping extension site-directed mutagenesis zg6pd M118-144 Gene:

[0031] A. Determine the 40th-48th amino acid of mutagenic zebrafish G6PD by comparing with human G6PD protein sequence. According to the zebrafish cDNA (NCBI Reference Sequence: XM_694076.5) sequence provided by NCBI, two pairs of primers were designed respectively. The upstream of the first pair of primers added BamHI restriction sites and ...

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Abstract

The invention discloses a transgenic zebrafish model with G6PD (gluocose-6-phosphate dehydrogenase deficiency) 118-144 site defects. The transgenic zebrafish model adopts an erythroid cell specific promoter gata-1 to drive a g6pd mutant gene with 118-144 site deletion and has EGFP (enhanced green fluorescent protein) expression traceability. The transgenic zebrafish model has the advantage that gata-1 is a specific transcription factor in original erythroid development and is used for labeling early erythroid cells. Establishment of blood circulation of zebrafish is started at 24 hpf, and the g6pd mutant gene can be expressed specifically in red blood cells when gata-1 is taken as the promoter to drive the g6pd mutant gene. The g6pd gene and an egfp gene are driven simultaneously, and traceable observation of expression of the g6pd mutant gene in the red blood cells can be realized. Whole bodies of a zebrafish embryo and an adult zebrafish are transparent, the constructed zebrafish model can trace drug-induced change rules of the fluorescence labeled red blood cells in the embryo and the fish body under a microscope, and a visible animal model is provided for screening drugs for treating G6PD deficiency.

Description

technical field [0001] The invention relates to a transgenic zebrafish model of G6PD118-144 site defect, belonging to the technical field of genetic engineering. Background technique [0002] Glucose-6-phosphate dehydrogenase deficiency (glucose-6-phosphate dehydrogenase deficiency, referred to as G6PD deficiency), commonly known as favism, is the most common hereditary hemolytic erythrocyte enzyme deficiency in the world. Red blood cells lose their nuclei and ribosomes during maturation, so they cannot synthesize proteins and cannot carry out the citric acid cycle. Therefore, mature red blood cells mainly rely on the pentose phosphate pathway to provide energy. G6PD is the rate-limiting enzyme of the pentose phosphate metabolic pathway. Glucose 6-phosphate is catalyzed by G6PD to produce nicotinamide adenine dinucleotide phosphate (NADPH) and ribose-5-phosphate (ribose-5-phosphate, R5P). NADPH is extremely important to maintain the reduced state of glutathione (GSH) to p...

Claims

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Application Information

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IPC IPC(8): A01K67/027A01K67/02C12N15/85C12N15/65
CPCA01K67/02A01K67/0275A01K2217/05A01K2227/40A01K2267/0306C12N15/65C12N15/8509C12N2800/106
Inventor 舒莉萍何志旭周艳华庹媛媛尚鲁俊吴西军崔冬冰
Owner GUIZHOU MEDICAL UNIV
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