Glycine N-methyltransferase (GNMT) Animal model and use thereof

a technology of glycine n-methyltransferase and animal model, which is applied in the field of glycine n-methyltransferase (gn-mt) animal model, can solve the problems of reducing the effectiveness of histological examination of tumors, no absolute methods for diagnosing or assessing the degree of malignancy of tumors, and cells may have lost their specific structural characteristics

Inactive Publication Date: 2009-06-11
NATIONAL YANG MING UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0082]The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.

Problems solved by technology

There are no absolute methods for diagnosing or assessing the degree of malignancy of tumors.
However, among the methods, microscopic examination of tissue is still the most reliable method for routine use.
However, on one hand, some cells may have lost their specific structural characters but still retain differentiated biochemical features, while others may still appear differentiated in structure but have lost many normal function attributes.
The two limitations reduce the effectiveness of histological examination of tumors.
In another aspect, such an examination by sampling specimens is not suitable for investigations on a large scale.
The lack of any absolute markers is a major deficiency in studying cancer.

Method used

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  • Glycine N-methyltransferase (GNMT) Animal model and use thereof
  • Glycine N-methyltransferase (GNMT) Animal model and use thereof
  • Glycine N-methyltransferase (GNMT) Animal model and use thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0083]To construct a targeting vector, DNA fragments digested from lambda phage clones 3-2 and 5-3 were inserted into a plasmid-pBluescrip II KS. Left arm was digested from the phage clone 5-3 by using Pst I and inserted into the pNeo vector. Right arm was digested from the phage clone 3-2 by using Hinc II and inserted into the TK vector. The fragment containing right arm and TK gene was digested by using Not I and inserted into the pNeo vector containing left arm to generate the targeting vector (FIG. 1).

[0084]The neomycin gene (to replace exons 1-4 and part of exon 5 of the mouse Gnmt gene) was framed with two DNA fragments (3.1 kb and 3.7 kb) in the targeting vector. The thymidine kinase gene was used as a negative selection marker (FIG. 2A). The 40 μg targeting vector was linearized using AscI and introduced into embryonic stem cells (129 / Sv-derived) by electroporation. After screening 278 clones using southern blot analysis (FIG. 2B), a recombinant clone was isolated and used f...

example 2

Materials and Methods:

Cell Culture and Treatment.

[0089]Hepatocellular carcinoma cell line [HA22T / VGH] was prepared according to Waxman, D. J. & O'Connor, C. Growth Hormone Regulation of Sex-Dependent Liver Gene Expression. Molecular Endocrinology 20, 2613 (2006), and the stable expression clones from human hepatoblastoma cell line-HepG2 was prepared based on Mode, A. & Gustafsson, J. A. Sex and the Liver—A Journey Through Five Decades. Drug Metabolism Reviews 38, 197-207 (2006), [SCG2] as stated in the paper Chen, S. Y. et al. Glycine N-methyltransferase tumor susceptibility gene in the benzo(a)pyrene-detoxification pathway. Cancer Res. 64, 3617-3623 (2004). were used in this example. Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (GIBCO BRL, Grand Island, N.Y.) containing 10% fetal bovine serum (Hyclone). AFB1 was solved in DMSO and treatment was performed in culture medium.

Immunofluorescent Staining and Confocal Microscopy.

[0090]Cultured HA22T / VGH cells were plac...

example 3

GNMT Nuclear Translocation is Induced by AFB1

[0101]GNMT cDNA transfected HA22T / VGH cells (FIGS. 10A and B) with AFB1 or DMSO (solvent control) for 16 hours. As shown in FIG. 10, GNMT distribution was initially restricted to the cytoplasm (FIG. 10A), but was partly translocated to cell nuclei following AFB1 treatment (FIG. 10B). These results showed that AFB1, as well as BaP, induces the nuclear translocation of GNMT.

[0102]It demonstrated that GNMT exhibited nuclear translocation in AFB1 treated cells (FIG. 10). It also showed that GNMT can reduce the formation of AFB1-DNA adducts and increase the survival rate of AFB1-treated cells. AFB1-DNA adducts formation have been implicated in liver carcinogenesis (Bressac, B. et al., Nature 350, 429-431, 1991; Hsu, I. C. et al., Nature 350, 427-428, 1991). It also proofed that the depletion of GNMT in hepatocyte raised the sensitivity of liver to this carcinogen. Given the choice, GNMT is involved in a cellular defense mechanism against thes...

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Abstract

The present invention is a new type of Glycine N-methyltransferase (GNMT) knockout mice model. This model can be applied to screen drug, test of treatment and search for diagnostic marker of hepatocellular carcinoma (HCC), glycogen storage disease, liver dysplasia, fatty liver and other liver disease.

Description

FIELD OF THE INVENTION[0001]The present invention relates to Glycine N-methyltransferase (GNMT) animal model and use thereof. The present invention also relates to the use of GNMT product in preventing or treating cancer, especially liver cancer.BACKGROUND OF THE INVENTION[0002]One of the most common types of human diseases throughout the world due to cell abnormalities is cancer, which is also the leading cause of death nowadays. Cancers are fully developed (malignant) tumors with a specific capacity to invade and destroy the underlying mesenchyme, i.e., local invasion. In some cases, invading tumor cells may further penetrate lymphatic vessels or blood vessels newly formed in the tumor and then may be carried to local lymph nodes or even to distant organs where they may produce secondary tumors (metastases). Tumors are usually recognized by the fact that the cells, which may arise from any tissue, are no longer responsive to at least some normal growth controlling mechanisms and h...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/45A61K31/7088A23L5/20
CPCA01K67/0276A01K2217/075C12N15/8509A01K2267/03A01K2267/0331A01K2227/105A61P1/16A61P3/08A61P35/00A61P39/02A61P43/00
Inventor CHEN, YI-MINGLIU, SHIH-PING
Owner NATIONAL YANG MING UNIVERSITY
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