Gene knockdown by large circular antisense for high-throughput functional genomics.
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.
Lee
Yun-Han
YH
WelGENE Inc., 71B 4L, Development Sector 2-3, Sungseo Industrial Park, Dalseogu, Daegu, 704-230, South Korea.
Moon
Ik-Jae
IJ
Hur
Bin
B
Park
Jeong-Hoh
JH
Han
Kil-Hwan
KH
Uhm
Seok-Yong
SY
Kim
Yong-Joo
YJ
Kang
Koo-Jeong
KJ
Park
Jong-Wook
JW
Seu
Young-Bae
YB
Kim
Young-Ho
YH
Park
Jong-Gu
JG
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*
D016373:DNA, Antisense; D009363:Neoplasm Proteins