title: Gene knockdown by large circular antisense for high-throughput functional genomics.
abstract: Single-stranded genomic DNA of recombinant M13 phages was tested as an antisense molecule and examined for its usefulness in high-throughput functional genomics. cDNA fragments of various genes (TNF-alpha, c-myc, c-myb, cdk2 and cdk4) were independently cloned into phagemid vectors. Using the life cycle of M13 bacteriophages, large circular (LC)-molecules, antisense to their respective genes, were prepared from the culture supernatant of bacterial transformants. LC-antisense molecules exhibited enhanced stability, target specificity and no need for target-site searches. High-throughput functional genomics was then attempted with an LC-antisense library, which was generated by using a phagemid vector that incorporated a unidirectional subtracted cDNA library derived from liver cancer tissue. We identified 56 genes involved in the growth of these cells. These results indicate that an antisense sequence as a part of single-stranded LC-genomic DNA of recombinant M13 phages exhibits effective antisense activity, and may have potential for high-throughput functional genomics.
authors.affiliation: WelGENE Inc., 71B 4L, Development Sector 2-3, Sungseo Industrial Park, Dalseogu, Daegu, 704-230, South Korea.
authors.forename: Yun-Han
authors.initials: YH
authors.lastname: Lee
authors.forename: Ik-Jae
authors.initials: IJ
authors.lastname: Moon
authors.forename: Bin
authors.initials: B
authors.lastname: Hur
authors.forename: Jeong-Hoh
authors.initials: JH
authors.lastname: Park
authors.forename: Kil-Hwan
authors.initials: KH
authors.lastname: Han
authors.forename: Seok-Yong
authors.initials: SY
authors.lastname: Uhm
authors.forename: Yong-Joo
authors.initials: YJ
authors.lastname: Kim
authors.forename: Koo-Jeong
authors.initials: KJ
authors.lastname: Kang
authors.forename: Jong-Wook
authors.initials: JW
authors.lastname: Park
authors.forename: Young-Bae
authors.initials: YB
authors.lastname: Seu
authors.forename: Young-Ho
authors.initials: YH
authors.lastname: Kim
authors.forename: Jong-Gu
authors.initials: JG
authors.lastname: Park
mesh_terms: D000818:Animals; D017104:Bacteriophage M13 / Q000235:genetics; D045744:Cell Line, Tumor; D002874:Chromosome Mapping / Q000379:methods*; D016373:DNA, Antisense / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D023281:Genomics / Q000379:methods*; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D016373:DNA, Antisense; D009363:Neoplasm Proteins