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Evaluation/screening method for diseases associated with D-amino acid utilizing Dao1-/-mouse

An evaluation method and amino acid technology, which can be used in the screening of compounds, botanical equipment and methods, biochemical equipment and methods, etc., and can solve the problem of unreported mating experiments.

Inactive Publication Date: 2011-07-13
KYUSHU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on mating experiments between DAO enzyme-deficient mice and other disease model mice

Method used

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  • Evaluation/screening method for diseases associated with D-amino acid utilizing Dao1-/-mouse
  • Evaluation/screening method for diseases associated with D-amino acid utilizing Dao1-/-mouse
  • Evaluation/screening method for diseases associated with D-amino acid utilizing Dao1-/-mouse

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Dao1 + / + , + / - and / or - / - Development of genotype determination method

[0062] Dao1 G181R It is a mutation in which guanine at position 661 in the nucleotide sequence of the cDNA of the Dao1 gene listed in SEQ ID NO: 3 is replaced with adenine. Therefore, in the wild type, it becomes the cleavage sequence (C↓CGG) of the restriction enzyme HpaII, whereas in Dao1 G181R In mutants, CCAG is not cut. Here, the nucleotides 625-726 including the mutation site are included in exon 7 (GenBank Accession No. NM_010018.2), so it can also be identified by the presence or absence of the HpaII cleavage site in chromosomal DNA wild type or mutant.

[0063] After individual identification of post-weaning mice, chromosomal DNA was extracted from the tail of each mouse individual using a commercially available mammalian genomic DNA miniprep kit (Miniprep Kit) (Sigma, G1N70-1 KT) and carried out. purification. As a forward primer, use the oligonucleotide consisting of the nucleo...

Embodiment 2

[0070] Development of Quantitative Analysis Method for Optical Isomers of Proline and 4-Hydroxyproline

[0071] Skin collagen contains a lot of proline and 4-hydroxyproline. Therefore, a method capable of simultaneously separating and quantitatively analyzing all optical isomers of both proline and 4-hydroxyproline has been developed.

[0072] Pic 4-1Represents the optical isomers of proline and 4-hydroxyproline. The optical isomers of proline are only L-form and D-form, but the optical isomers of 4-hydroxyproline are trans and cis in addition to the difference between L-form and D-form. There are 4 types in total. First, amino acids are derivatized with fluorescent reagent NBD-F for fluorescent labeling. Then, if Figure 4-2 As shown, reversed-phase separation chromatography was performed on the first column to detect the peaks of trans-4-hydroxyproline, cis-4-hydroxyproline and proline. Then, fractions of the aforementioned peaks were collected by a column switching v...

Embodiment 3

[0077] Tumor proliferation in Dao1 enzyme-deficient mice

[0078] DMEM (Sigma) medium supplemented with 10% fetal calf serum (Irvine Scientific, batch #300A80601) was used in 5% CO 2 1. The sarcoma cells of the Swiss Webster Sarcoma 180 strain were cultured under humidified conditions at 37°C. modulation 1x10 7 0.05 mL of the suspension per mL was transplanted into the right hind paw pad of Dao1 enzyme-deficient mice or wild-type mice by intradermal injection. The long diameter, short diameter and thickness of the tumor were measured with a vernier caliper every week after transplantation, and the tumor volume was calculated by the following formula.

[0079] Tumor volume (mm 3 ) = long diameter (mm) x short diameter (mm) x (thickness (mm)-3)

[0080] The thickness here assumes that the original foot thickness is 3 mm, and the difference from this 3 mm is used as the thickness of the tumor.

[0081] The result is as Figure 8 shown. Figure 8 It is a bar graph comparing...

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Abstract

Disclosed is an evaluation method which can select a Dao- / - homozygote rapidly among many animals produced by the mating experiment between a DAO oxygen-defect mouse and another disease model mouse to quantify a D-amino acid contained in many samples rapidly. Specifically disclosed is a method for evaluating the influence of test conditions on a mouse biological tissue or a cultured tissue cell derived from the biological tissue. The method comprises the steps of: providing a Dao1- / - mouse or the like; exposing a biological tissue from the Dao1- / - mouse or the like, or the like, to the test conditions; and analyzing the influence of the exposure of the biological tissue from the Dao1- / - mouse or the like, or the like, to the test conditions.

Description

technical field [0001] The present invention relates to a method for evaluating the effects of test conditions on mouse living tissue or cultured tissue cells derived from the living tissue, an evaluation system for implementing the evaluation and screening method, and pharmaceuticals and / or cosmetics using the evaluation system Candidate substance. Background technique [0002] All amino acids except glycine have two optical isomers, D form and L form. L-amino acids are used in biological protein synthesis, and most of the amino acids contained in proteins are L-amino acids. In contrast, D-amino acids exist in some physiologically active peptides of lower organisms, and many of them are biosynthesized through the process of post-translational modification. Therefore, amino acids constituting proteins or peptides are mainly L-amino acids, with exceptions of D-amino acids. [0003] D-amino acid is a constituent of peptidoglycan in the cell wall of bacteria. In addition, f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00C12N15/09C12Q1/02C12Q1/68G01N30/88G01N33/15G01N33/50G01N33/53G01N33/577
CPCA01K67/0276G01N33/5088C12N9/0024G01N2333/90644C12Q1/26C12N15/8509A01K2267/0356C07K14/4702B01J20/29A01K2217/075A01K2227/105A01K2217/15A01K2267/0331G01N2500/10A01K2217/03
Inventor 浜濑健司财津洁三田真史芦田丰东条洋介
Owner KYUSHU UNIV
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