Identification of Genes Involved in Liver Cancer Cell Growth Using an Antisense Library of Phage Genomic DNA
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Purpose: Genes involved in liver cancer cell growth have been identified using an antisense library of large circular (LC-) genomic DNA of a recombinant M13 phage. Materials and Methods: A subtracted cDNA library was constructed by combining procedures of suppression subtractive hybridization (SSH) and unidirectional cloning of the subtracted cDNA into an M13 phagemid vector. Utilizing the life cycle of M13 bacteriophages, LC-antisense molecules derived from 1,200 random cDNA clones selected by size were prepared from the culture supernatant of bacterial transformants. The antisense molecules were arrayed for transfection on 96-well plates preseeded with HepG2. Results: When examined for growth inhibition after antisense transfection, 153 out of 1,200 LC-antisense molecules showed varying degrees of growth inhibitory effect to HepG2 cells. Sequence comparison of the 153 clones identified 58 unique genes. The observations were further extended by other cell-based assays. Conclusion: These results suggest that the LC-antisense library offers potential for unique high-throughput screening to find genes involved in a specific biological function, and may prove to be an effective target validation system for gene-based drug discovery. (Cancer Res Treat. 2004;36:246-254)
Abstract
Purpose: Genes involved in liver cancer cell growth have been identified using an antisense library of large circular (LC-) genomic DNA of a recombinant M13 phage. Materials and Methods: A subtracted cDNA library was constructed by combining procedures of suppression subtractive hybridization (SSH) and unidirectional cloning of the subtracted cDNA into an M13 phagemid vector. Utilizing the life cycle of M13 bacteriophages, LC-antisense molecules derived from 1,200 random cDNA clones selected by size were prepared from the culture supernatant of bacterial transformants. The antisense molecules were arrayed for transfection on 96-well plates preseeded with HepG2. Results: When examined for growth inhibition after antisense transfection, 153 out of 1,200 LC-antisense molecules showed varying degrees of growth inhibitory effect to HepG2 cells. Sequence comparison of the 153 clones identified 58 unique genes. The observations were further extended by other cell-based assays. Conclusion: These results suggest that the LC-antisense library offers potential for unique high-throughput screening to find genes involved in a specific biological function, and may prove to be an effective target validation system for gene-based drug discovery. (Cancer Res Treat. 2004;36:246-254)
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