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Transformed caenorhabditis elegans and method for screening for substances regulating glucose metabolism using same

A Caenorhabditis elegans, sugar metabolism technology, applied in the direction of using vectors to introduce foreign genetic material, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., to achieve efficient research results

Inactive Publication Date: 2014-02-05
POSTECH ACAD IND FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there has been no study using C. elegans to screen candidate genes and drug candidates involved in glucose metabolism and diabetes

Method used

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  • Transformed caenorhabditis elegans and method for screening for substances regulating glucose metabolism using same
  • Transformed caenorhabditis elegans and method for screening for substances regulating glucose metabolism using same
  • Transformed caenorhabditis elegans and method for screening for substances regulating glucose metabolism using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Preparation method of transformed Caenorhabditis elegans-DNA integration

[0038] Since the far-3::GFP DNA inserted into C. elegans is of the type not contained in the chromosome, the far-3::GFP DNA will not be transmitted to the progeny of C. elegans. For experiments using such transformed C. elegans, screening of transformed C. elegans is difficult and the expression of far-3::GFP is not uniform.

[0039] Therefore, in order to obtain stably transformed C. elegans, the DNA was altered by UV irradiation, exogenous far-3::GFP DNA was introduced and integrated into the chromosome. DNA integration by UV integration proceeds with a low probability, so the success rate of obtaining large numbers of C. elegans by UV radiation may increase.

[0040]The transformation of C. elegans can be identified under light microscopy by inserting the gene obtained from the short body mutant (dpy-5) together with far-3::GFP DNA into the long body. During DNA integration, trans...

Embodiment 2

[0042] Example 2. Measurement of fluorescence of far-3::GFP transformed C. elegans in the presence of glucose

[0043] The far-3::GFP-transformed C. elegans prepared in Example 1 was grown in a medium containing 2% glucose, and the fluorescence was measured by the following method.

[0044] Specifically, a drop of 2% agarose was dropped on a glass slide, and another glass slide was covered to spread the agarose thinly therein. Remove the glass slide after the agarose has hardened to form a plate, and place a drop of NaN at a concentration of 50mM 3 Drop onto an agarose plate, and then transfer about 15 C. elegans on it to anesthetize. Carefully cover the coverslip without air bubbles and observe the fluorescence of far-3::GFP by taking images using a fluorescence microscope. A Zeiss Axio Scope A1 was used as a fluorescence microscope. The fluorescence displayed by the whole body of C. elegans was quantitatively analyzed using ImageJ software, and the fluorescence of more th...

Embodiment 3

[0046] Embodiment 3. Use RNAi to find the method for sugar metabolism gene

[0047] The inventors of the present invention used the Julie Ahringer RNAi bacterial library to suppress the expression of specific genes and observed the fluorescence of far-3::GFP. Bacteria obtained from the library were streaked and transferred to LB / ampicillin plates for 12 to 16 hours at 37°C in an incubator. One bacterial colony growing on the plate was selected, transferred to liquid LB / ampicillin medium, and incubated at 37°C for 12 hours in a shaking incubator. After 12 hours, 100 μl of LB / ampicillin medium grown with bacteria was dispensed into NG medium including 2% glucose and ampicillin, and bacteria were cultured at 37° C. for 12 to 16 hours. To induce the reaction, 50 μl of isopropyl-1-thio-β-D-galactopyranoside (IPTG) at a concentration of 100 mM was added to the bacteria, reacted at room temperature for one day, and two adult far-3 ::GFP-transformed Caenorhabditis elegans was transf...

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Abstract

The present invention relates to a method for preparing a transformed Caenorhabditis elegans (C. elegans) which reacts to glucose by exhibiting fluorescence, and to a method for screening for a candidate substance and for a novel gene capable of regulating glucose metabolism and metabolic diseases using the transformed Caenorhabditis elegans. The transformed Caenorhabditis elegans of the present invention is prepared such that changes in glucose may be easily observed on a real-time basis using a fluorescent microscope. The transformed Caenorhabditis elegans of the present invention enables substances for regulating glucose metabolism to be quickly and accurately discovered, and further, may be expected to significantly contribute to studies on a variety of incurable metabolism-related diseases such as obesity, diabetes, and the like.

Description

technical field [0001] The present invention relates to a method for preparing transformed Caenorhabditis elegans (abbreviated as C. elegans) that exhibits fluorescence in response to glucose, and a method for screening candidate substances and new genes using the Caenorhabditis elegans, the substances and new Genes can regulate glucose metabolism and metabolic diseases. Background technique [0002] Because of its easy gene regulation and small size, C. elegans can be grown in large quantities in vitro at a relatively low cost. C. elegans is suitable for experimental applications because it only takes about 3 days after egg hatching to pass through four stages L1, L2, L3 and L4 to become an adult. Caenorhabditis elegans has a simple structure, excluding germ cells, only 959 cells, and it is transparent. For these reasons, it is easy to directly observe the interior of C. elegans through a microscope. [0003] Furthermore, cell lines from embryos to adults have been fully...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C12N5/10C12N15/62C12N15/85C12N15/63
CPCA01K67/0336C12N2015/8527A01K2217/052A01K2267/0393C12N15/62C12N15/85A01K67/027G01N33/5085
Inventor 李昇宰李东烨
Owner POSTECH ACAD IND FOUND
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