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Gene knockdown by large circular antisense for high-throughput functional genomics

Nature Biotechnology volume 23, pages 591–599 (2005)Cite this article

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-α, 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.

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Figure 1: Stability and antisense activity of LC-antisense.
Figure 2: Target specificity and antisense activity of LC-antisense.
Figure 3: High-throughput functional analysis to identify genes involved in the growth of liver cancer cells.
Figure 4: Effects of LC-antisense on cell cycle progression and apoptotic induction.
Figure 5: Validation of WGSL 11 as a potential target for the inhibition of cancer cell growth.

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Acknowledgements

This study was supported by generous grants of the CDRC of Korean Science & Engineering Foundation (research grant no. R01-2000-00138, R13-2002-028-01004-0), South Korea, and WelGENE Inc., a biotechnology company founded by Jong-Gu Park.

Author information

Author notes

  1. Yun-Han Lee, Ik-Jae Moon and Bin Hur: These authors contributed equally to this work.

Authors and Affiliations

  1. WelGENE Inc., 71B 4L, Development Sector 2-3, Sungseo Industrial Park, Dalseogu, Daegu, 704-230, South Korea

    Yun-Han Lee, Ik-Jae Moon, Bin Hur, Jeong-Hoh Park, Kil-Hwan Han, Seok-Yong Uhm, Yong-Joo Kim & Jong-Gu Park

  2. Department of General Surgery, Dongsan Medical Center, Keimyung University, Kyungpook National University, 1370 Sangyeokdong, Bookgu, Daegu, 702-701, South Korea

    Koo-Jeong Kang

  3. Department of Immunology, Keimyung University School of Medicine, Kyungpook National University, 1370 Sangyeokdong, Bookgu, Daegu, 702-701, South Korea

    Jong-Wook Park

  4. Department of Microbiology, College of Natural Sciences, Kyungpook National University, 1370 Sangyeokdong, Bookgu, Daegu, 702-701, South Korea

    Yun-Han Lee, Young-Bae Seu & Young-Ho Kim

  5. Department of Medical Genetic Engineering, Keimyung University School of Medicine, Dongsan Medical Center, 194 Dongsandong, Joonggu, Daegu, 700-712, South Korea

    Jong-Gu Park

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Supplementary information

Supplementary Fig. 1

A schematic diagram for the construction of the phage genomic LC-antisense molecule for rat TNF-α (TNFα -LCAS). (PDF 113 kb)

Supplementary Fig. 2

Effect of cdk2-LCAS on the CDK2 protein level. (PDF 83 kb)

Supplementary Fig. 3

Real-time RT-PCR analysis of target gene expression in HeLa cells treated with cdk2- / cdk4-LCAS or cdk2 siRNA. (PDF 122 kb)

Supplementary Fig. 4

Effect of cdk4-LCAS on the CDK4 protein level. Western blot analysis was performed for the CDK4 expression in MCF7 cells 48 h after the treatments of cdk4-LCAS and cdk4-LCSE molecules. (PDF 73 kb)

Supplementary Fig. 5

A schematic diagram for the construction of a random-gene LC-antisense library. (PDF 99 kb)

Supplementary Table 1

List of cloning primers and sizes (PDF 72 kb)

Supplementary Table 2

List of RT-PCR primers (PDF 65 kb)

Supplementary Table 3

List of primers for real-time quantitative RT-PCR (PDF 58 kb)

Supplementary Table 4

List of PS end capped AS-oligos used for the antisense effect on WGSL11 expression. (PDF 46 kb)

Supplementary Methods (PDF 127 kb)

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Lee, YH., Moon, IJ., Hur, B. et al. Gene knockdown by large circular antisense for high-throughput functional genomics. Nat Biotechnol 23, 591–599 (2005). https://doi.org/10.1038/nbt1089

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