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  • Review Article
  • Published:

Improving the efficiency of RNA interference in mammals

Nature Reviews Genetics volume 5, pages 355–365 (2004)Cite this article

Key Points

  • RNA interference (RNAi) is the process in which dsRNA leads to gene silencing, by either inducing the sequence-specific degradation of complementary mRNA or inhibiting translation.

  • RNAi has been successfully applied as a powerful gene-silencing approach to various organisms, including Caenorhabditis elegans, plants, Drosophila melanogaster and mouse oocytes.

  • The use of long dsRNAs in mammalian systems has been limited primarily because the introduction of dsRNA that is longer than 30 nucleotides (nt) induces a nonspecific interferon response. However, short 21–22-nt dsRNA molecules — known as small interfering RNAs (siRNAs) — could be used to target mammalian genes by RNAi while evading the interferon response.

  • RNAi in mammals is triggered by various types of molecule, including synthetic siRNAs, plasmid-based short hairpin RNAs (shRNAs) or endogenous hairpin micro RNAs (miRNAs).

  • It is now possible to carry out gene-silencing experiments in various cell types and cell lines, as well as in living animals. This is largely due to the development of DNA-vector-based siRNA-expression systems that allow the stable and prolonged silencing of target genes, together with a wider choice of delivery methods. Germline transmission of cells that contain an shRNA transgene — such as embryonic stem cells in mice and fertilized eggs in rats — has also been accomplished.

  • The specificity and efficiency of RNAi in mammals has improved greatly thanks to advances in rational design, the ability to screen for the most effective siRNAs and tools that allow the inducible suppression of endogenous genes.

  • Genome-wide functional RNAi screens, which were previously carried out exclusively in worms and flies, have now begun to revolutionize large-scale loss-of-function studies in mammals.

  • The application of RNAi in mammals has the potential to allow the systematic analysis of gene expression and holds the promise of therapeutic gene silencing.

Abstract

RNA interference (RNAi) has been very successfully applied as a gene-silencing technology in both plants and invertebrates, but many practical obstacles need to be overcome before it becomes viable in mammalian systems. Greater specificity and efficiency of RNAi in mammals is being achieved by improving the design and selection of small interfering RNAs (siRNAs), by increasing the efficacy of their delivery to cells and organisms, and by engineering their conditional expression. Genome-wide functional RNAi screens, which are predominantly done in worms and flies, have now begun to revolutionize large-scale loss-of-function studies in mammals.

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Figure 1: RNAi-mediated gene silencing in mammals.
Figure 2: The generation of effective siRNA.
Figure 3: Microarray-based screening for effective siRNA.
Figure 4: Large-scale RNAi screens in cells.

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Acknowledgements

The author thanks the members of his laboratory for critical reading of the manuscript and several other investigators for sharing their unpublished work. We acknowledge that space limitations might have precluded the citation of work of some investigators. The author was supported by grants from Cold Spring Harbor Laboratory and the National Institute of Health.

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  1. Cancer Genome Research Center, Cold Spring Harbor Laboratory, 500 Sunnyside Boulevard, Woodbury, 11797, New York, USA

    Vivek Mittal

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DATABASES

LocusLink

AKT1

CCR5

CD44

CXCR4

cyclophilin B

CYLD

Dicer

DNMT1

FRA1

Hh

Id

IGF1R

LMNA

LMO2

MyoD

PLK1

RB

SOD1

TH

TP53

OMIM

X-SCID

FURTHER INFORMATION

Vivek Mittal's laboratory

Nature Reviews web focus on RNAi

NCBI Unigene

RNAi animation

Glossary

INTERFERONS

A group of glycoprotein cytokines that are produced by animal cells when they are invaded by viruses. They are also activated by long dsRNA, which results in the activation of 2′–5′ oligoadenylate synthase and protein kinase PKR. The 2′–5′ oligonucleotide activates RNAseL, which leads to mRNA cleavage. PKR phosphorylates the translation initiation factor eI2α, which leads to global inhibition of mRNA translation.

HOMOLOGOUS RECOMBINATION

The process of replacing an endogenous gene with an artifical cassette that is flanked by regions that are homologous to sequences bordering the targeted gene. It is used as a technique to inactivate a gene and determine its function in a living animal.

ANTISENSE VECTORS

Vectors that express DNA or RNA molecules that are complementary to sequences on target mRNA and result in the inhibition of protein synthesis.

FEATURE

A spot on a microarray that has a defined shape, size and intensity.

ORTHOTOPIC

The transplantation of foreign tumour cells into another species at its normal anatomical position.

TRAIL-INDUCED APOPTOSIS

The tumour necrosis factor (TNF)-receptor-related apoptosis-inducing ligand (TRAIL) is a secreted protein ligand that binds to its receptor and induces apoptosis selectively in tumour cells but not in normal cells.

SYNTHETIC LETHAL MUTATIONS

Two mutations are considered to be synthetically lethal if in combination they result in cell death, whereas either alone leads to a viable cell.

BARCODE

A short stretch of DNA that functions as a unique molecular fingerprint for each short hairpin RNA in a library. The molecular barcode can be identified and recovered by PCR amplification, by using primers that are specific to each barcode.

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Mittal, V. Improving the efficiency of RNA interference in mammals. Nat Rev Genet 5, 355–365 (2004). https://doi.org/10.1038/nrg1323

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