DOI: 10.1073/pnas.2136685100. An approach to genomewide screens of expressed small interfering RNAs in mammalian cells

An approach to genomewide screens of expressed small interfering RNAs in mammalian cells

10.1073/pnas.2136685100

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

To facilitate the construction of large genomewide libraries of small interfering RNAs (siRNAs), we have developed a dual promoter system (pDual) in which a synthetic DNA encoding a gene-specific siRNA sequence is inserted between two different opposing polymerase III promoters, the mouse U6 and human H1 promoters. Upon transfection into mammalian cells, the sense and antisense strands of the duplex are transcribed by these two opposing promoters from the same template, resulting in a siRNA duplex with a uridine overhang on each 3′ terminus. A single-step PCR protocol has been developed by using this dual promoter system that allows the production of siRNA expression cassettes in a high-throughput manner. We have shown that siRNAs transcribed by either the dual promoter vector or siRNA expression cassettes can induce strong and gene-specific suppression of both endogenous genes and ectopically expressed genes in mammalian cells. Furthermore, we have constructed an arrayed siRNA expression cassette library that targets >8,000 genes with two siRNA sequences per gene. A high-throughput screen of this library has revealed both known and unique genes involved in the NF-κB signaling pathway.

Bibliography

Zheng, L., Liu, J., Batalov, S., Zhou, D., Orth, A., Ding, S., & Schultz, P. G. (2003). An approach to genomewide screens of expressed small interfering RNAs in mammalian cells. Proceedings of the National Academy of Sciences, 101(1), 135â140.

Authors 7

Lianxing Zheng (first)
Jun Liu (additional)
Sergei Batalov (additional)
Demin Zhou (additional)
Anthony Orth (additional)
Sheng Ding (additional)
Peter G. Schultz (additional)

References 33 Referenced 154

Dates

Type	When
Created	21 years, 7 months ago (Jan. 6, 2004, 1:12 p.m.)
Deposited	3 years, 4 months ago (April 25, 2022, 10:20 p.m.)
Indexed	2 months, 2 weeks ago (June 18, 2025, 3:50 a.m.)
Issued	21 years, 8 months ago (Dec. 19, 2003)
Published	21 years, 8 months ago (Dec. 19, 2003)
Published Online	21 years, 8 months ago (Dec. 19, 2003)
Published Print	21 years, 7 months ago (Jan. 6, 2004)

Funders 0

None

BibTeX

@article{Zheng_2003, title={An approach to genomewide screens of expressed small interfering RNAs in mammalian cells}, volume={101}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.2136685100}, DOI={10.1073/pnas.2136685100}, number={1}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Zheng, Lianxing and Liu, Jun and Batalov, Sergei and Zhou, Demin and Orth, Anthony and Ding, Sheng and Schultz, Peter G.}, year={2003}, month=dec, pages={135–140} }

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