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Transgenic zebra fish model with g6pd1303-1497 locus deletion and construction method

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

Active Publication Date: 2016-10-26
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 zebra fish model with g6pd1303-1497 locus deletion and construction method
  • Transgenic zebra fish model with g6pd1303-1497 locus deletion and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The method of constructing genetic zebrafish model includes the following steps:

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

[0024] G6pd M1303-1497 -EGFP-PCS 2+ The two plasmids of gata-1-PBSK-Isce I are recombined according to the following connection system:

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

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

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

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

[0029] 3) Put the mixed competent E. coli suspension in a water bath at 42°C for 90 seconds and place it on ice quickly for 90 seconds;

[0030] 4) Add 300μl of LB liquid cultu...

Embodiment 2

[0061] An application of a transgenic zebrafish model. The transgenic zebrafish model obtained in Example 1 is used in the process of screening drugs for the treatment of G6PD deficiency, and changes in red blood cells are observed during drug treatment. The transgenic zebrafish model is a transgenic model with 1303-1497 point mutation G6PD gene, which encodes the amino acid sequence of 435-499 in the zebrafish G6PD protein sequence, which is higher than the amino acid sequence of 447-489 in the human G6PD protein sequence. Conservative. The amino acid sequence 447-489 in the human G6PD protein sequence is the NADP+ binding site, 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 Sexual and functional integrity. gata1-g6pd M1303-1497 -EGFP transgenic zebrafish embryos and juveniles are shown in Figure 1. The zebrafish embryos and...

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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, and particularly relates to a transgenic zebrafish model with g6pd1303-1497 deletion and a construction method. Background technique [0002] Glucose-6-phosphate dehydrogenase deficiency (gluocose-6-phosphate dehydrogenase deficiency, G6PD deficiency for short), commonly known as fava bean disease, is the most common hereditary hemolytic erythrocyte enzyme deficiency in the world. G6PD gene mutation leads to the decrease of G6PD enzyme activity, which mainly affects the enzyme activity through two mechanisms, namely, the effect of the G6PD functional structure region and the formation of G6PD dimers. G6PD enzyme is the initial rate-limiting enzyme involved in the red blood cell glycolysis pentose phosphate oxidation pathway. Its main function is to catalyze the production of an important reducing substance reduced coenzyme II (NADPH), and NADPH is an important antioxidant substance ...

Claims

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Application Information

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IPC IPC(8): A01K67/027A01K67/02C12N15/85C12N15/65A61K49/00
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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