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.

Spin-Lattice Relaxation Time in Drug Discovery and Design

“…These unannotated proteins may provide important information for the development of new antimicrobial agents, especially because many of them are not identified in mammals, thus being potential targets for selective chemotherapy. Usually, the biological importance and relevance of proteins can be determined by methods such as gene knockout or RNA interference (RNAi), however, those approaches are not effective to determine the protein function [20]. For that last purpose, complete protein sequence similarity processes are used, but they could lead to errors and do not guarantee the determination of the specific function of one of these molecules.…”

Fabrication of a microarray using a combination of the large circular sense and antisense DNA

“…Either pSPORT1 or pT3T7-Pac phagemids, harboring human EST cDNA, were utilized in the production of single-stranded phage genomic DNA harboring the sense cDNA sequences. The recombinant phagemids were transformed into competent E. coli cells, XL-1 Blue (Stratagene, La Jolla, CA, USA), which had been infected with the helper bacteriophage, M13K07 (New England Biolabs, Ipswich, MA, USA), in accordance with a previously described method (20,21). All of the LC-sense DNA was adjusted to a uniform concentration of 0.35 g/l.…”

“…The M13 phagemid, a recombinant plasmid vector which is used for the construction of recombinant bacteriophages, can be engineered to generate a large quantity of single-stranded large circular (LC) molecules harboring either the sense or antisense sequence of a gene. Recently, high-throughput functional genomics using LC-antisense molecules has been developed for the identification of genes associated with cancer cell growth (20). The LC-sense DNA of recombinant bacteriophages may result in advantages for higher chances of binding to complementary target cDNA, owing to its considerable length and high degree of sequence fidelity.…”

Integrating Experimental and Analytic Approaches to Improve Data Quality in Genome-wide RNAi Screens

“…14, 39 An additional consideration is that the positive controls should be as effective as the hits in the screen are expected to be. An example of this is shown in the HCV screens.…”