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Related Experiment Videos

Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells.

S Yang1, S Tutton, E Pierce

  • 1Department of Dermatology and Cutaneous Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, and Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.

Molecular and Cellular Biology
|October 18, 2001
PubMed
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RNA interference (RNAi) effectively suppresses gene expression in many organisms. In mammalian cells, long double-stranded RNA (dsRNA) can induce sequence-specific gene silencing in undifferentiated embryonic stem cells, but this effect is transient.

Area of Science:

  • Molecular Biology
  • Gene Expression Regulation
  • RNA Interference

Background:

  • RNA interference (RNAi) is a potent gene silencing mechanism observed in various organisms, but its application in mammalian systems remains limited.
  • Investigating RNAi feasibility in mammalian cells is crucial for understanding gene regulation and developing therapeutic strategies.

Purpose of the Study:

  • To explore the potential of RNA interference (RNAi) for gene silencing in mammalian cells.
  • To assess the efficacy of double-stranded RNA (dsRNA) in suppressing gene expression in different mammalian cell types, including embryonic stem (ES) cells.

Main Methods:

  • Utilized the enhanced green fluorescent protein (eGFP) gene as a target for RNAi.
  • Introduced dsRNA into mammalian cells via plasmid transfection (in situ dsRNA production) or direct transfection of in vitro transcribed dsRNA.

Related Experiment Videos

  • Tested RNAi in various mammalian cells, including differentiated and undifferentiated ES cells.

    • Main Results:

    • Transient transfection with long dsRNA did not induce specific RNAi in most tested mammalian cells, including differentiated ES cells.
    • Sequence-specific RNAi was achieved in undifferentiated ES cells using long dsRNA, reducing target gene expression by up to 70% at 8.3 nM.
    • RNAi effects were transient, diminishing after 5 days, suggesting potential masking by the interferon response in mammalian cells.
    • Mammalian cell cytoplasmic extracts processed long dsRNA into small RNAs (21-22 nucleotides), indicating the presence of RNAi machinery.

    Conclusions:

    • Mammalian cells possess RNAi machinery, but the non-specific interferon response to long dsRNA may impede observable sequence-specific gene silencing.
    • Undifferentiated ES cells exhibit reduced interferon response, making them more amenable to RNAi-mediated gene silencing.
    • This study highlights the potential of dsRNA for transient gene expression inhibition in ES cells to investigate differentiation processes.