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Transgenic zebrafish model with deletion of g6pd1303-1497 and its construction method

A zebrafish, transgenic technology, applied in genetic engineering, biochemical equipment and methods, pharmaceutical formulations, etc.

Active Publication Date: 2019-10-29
GUIZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is still no specific treatment for G6PD deficiency at home and abroad, and there is no ideal animal experimental model that can well realize the research on the relationship between the structural changes and functions of the G6PD gene

Method used

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  • Transgenic zebrafish model with deletion of g6pd1303-1497 and its construction method
  • Transgenic zebrafish model with deletion of g6pd1303-1497 and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The construction method of gene zebrafish model comprises the steps:

[0023] (1) Build gata1-g6pd M1303-1497 -EGFP-PBSK-IsceI recombinant plasmid:

[0024] will g6pd M1303-1497 -EGFP-PCS 2+ , two plasmids of gata-1-PBSK-Isce I are recombined and connected according to the following connection system:

[0025] A.g6pd M1303-1497 -EGFP-PCS 2+ Plasmid 3 μL, gata-1-PBSK-Isce I plasmid 5 μL, 10×T4DNA Ligase Buffer 1 μL, T4DNA Ligase 1 μL, total volume 10 μL. After mixing well, store at 4°C.

[0026] B. The recombined gata1-g6pd M1303-1497 -EGFP-PBSK-Isce I plasmid transformation and screening of monoclonal colonies:

[0027] 1) Thaw a competent E. coli suspension on ice;

[0028] 2) Add the target ligation product into the competent E. coli suspension with a pipette, blow gently until mixed, and place on ice for 30 minutes;

[0029] 3) Place the mixed competent Escherichia coli suspension in a water bath at 42°C for 90 seconds, then quickly place it on ice for 90 s...

Embodiment 2

[0061] An application of a transgenic zebrafish model. The transgenic zebrafish model obtained in Example 1 was used to track and observe changes in red blood cells during drug treatment during the process of screening drugs for the treatment of G6PD deficiency. The transgenic zebrafish model is a transgenic model in which the 1303-1497 site mutation G6PD gene is transferred. This gene encodes the 435-499 amino acid sequence in the zebrafish G6PD protein sequence, which is highly similar to the 447-489 amino acid sequence in the human G6PD protein sequence. keep. The 447-489 amino acid sequence in the human G6PD protein sequence is the binding site of NADP+, which contains two G6PD gene mutation sites, G1376T and G1388A, which are common in Chinese G6PD deficiency. The integrity of this amino acid sequence directly affects the structural integrity of G6PD integrity and functional integrity. gata1-g6pd M1303-1497 -EGFP transgenic zebrafish embryos and juveniles are shown in F...

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Abstract

The invention discloses a transgenic zebra fish model with g6pd1303-1497 locus deletion. gatal-g6pdM1303-1497-EGFP-PBSK-Isce I plasmids with g6pd1303-1497 locus deletion of g6pd gene and Isce I enzyme are injected into embryo during a unicell stage. The advantages of the transgenic zebra fish model with g6pd1303-1497 locus deletion are that the g6pd gene and an EGFP gene can be driven at the same time, tracking observation of expression in red blood cells of the mutant type g6pd gene with the 1303-1497 locus deletion can be achieved.

Description

technical field [0001] The invention belongs to the technical field of design genetic engineering, in particular to a transgenic zebrafish model with g6pd1303-1497 site deletion and a construction method. 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. Mutations in the G6PD gene lead to a decrease in the enzyme activity of G6PD, which mainly affects the activity of the enzyme through two mechanisms, that is, affecting the function of the functional structural region of G6PD and the formation of G6PD dimers. G6PD enzyme is the initial rate-limiting enzyme involved in the erythrocyte glycolytic pentose phosphate oxidation pathway. Its main function is to catalyze the formation of an important reducing substance, reduced coenzyme II (NADPH), and NADPH is an importa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85A01K67/027A01K67/02C12N15/65A61K49/00
CPCA01K67/02A01K67/0275A01K2217/05A01K2227/40A01K2267/0306A61K49/0008C12N15/65C12N15/8509C12N2800/106
Inventor 舒莉萍何志旭吴西军周艳华夏海雄宋锦庹媛媛尚鲁俊
Owner GUIZHOU MEDICAL UNIV
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