Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells.
OBJECTIVE
To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells.


METHODS
We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products.


RESULTS
Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h.


CONCLUSIONS
Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.
Li
Yong
Y
Beijing Institute for Cancer Research, Beijing Laboratory of Molecular Oncology, School of Oncology, Peking University, 1 Da-Hong-Luo-Chang Street, Western District, Beijing 100034, China.
Lu
You-Yong
YY
D048868:Adaptor Proteins, Signal Transducing; D000498:Allyl Compounds / Q000494:pharmacology*; D016564:Amyloid beta-Protein Precursor; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D020224:Expressed Sequence Tags; D005737:Garlic / Q000737:chemistry*; D015972:Gene Expression Regulation, Neoplastic; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D015849:Plasminogen Inactivators / Q000235:genetics / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D017422:Sequence Analysis, DNA / Q000379:methods; D058258:Serpin E2; D013274:Stomach Neoplasms / Q000235:genetics*; D013440:Sulfides / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D000498:Allyl Compounds; D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; C110719:LAMTOR5 protein, human; D015849:Plasminogen Inactivators; D010975:Platelet Aggregation Inhibitors; D058255:Protease Nexins; D011956:Receptors, Cell Surface; C545486:SERPINE2 protein, human; D058258:Serpin E2; D013440:Sulfides; D017361:Viral Nonstructural Proteins; C042577:diallyl trisulfide