Identification of Genes Involved in Liver Cancer Cell Growth Using an Antisense Library of Phage Genomic DNA

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 M 13 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. S equence 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.