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PLoS One. 2014 May 15;9(5):e96445. doi: 10.1371/journal.pone.0096445. eCollection 2014.

High-throughput screening of effective siRNAs using luciferase-linked chimeric mRNA.

Author information

1
Department of Orthopaedic Surgery, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
2
UCLA School of Nursing, Division of Hematology-Oncology, David Geffen School of Medicine at UCLA and UCLA AIDS Institute, Los Angeles, California, United States of America.
3
Department of Molecular Genetics and Medicine and AIDS Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
4
School of Dentistry, University of California Los Angeles, Los Angeles, California, United States of America; Department of Orthodontics, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, China.

Abstract

The use of siRNAs to knock down gene expression can potentially be an approach to treat various diseases. To avoid siRNA toxicity the less transcriptionally active H1 pol III promoter, rather than the U6 promoter, was proposed for siRNA expression. To identify highly efficacious siRNA sequences, extensive screening is required, since current computer programs may not render ideal results. Here, we used CCR5 gene silencing as a model to investigate a rapid and efficient screening approach. We constructed a chimeric luciferase-CCR5 gene for high-throughput screening of siRNA libraries. After screening approximately 900 shRNA clones, 12 siRNA sequences were identified. Sequence analysis demonstrated that most (11 of the 12 sequences) of these siRNAs did not match those identified by available siRNA prediction algorithms. Significant inhibition of CCR5 in a T-lymphocyte cell line and primary T cells by these identified siRNAs was confirmed using the siRNA lentiviral vectors to infect these cells. The inhibition of CCR5 expression significantly protected cells from R5 HIV-1JRCSF infection. These results indicated that the high-throughput screening method allows efficient identification of siRNA sequences to inhibit the target genes at low levels of expression.

PMID:
24831610
PMCID:
PMC4022502
DOI:
10.1371/journal.pone.0096445
[Indexed for MEDLINE]
Free PMC Article

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