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Related Experiment Videos

Establishing efficient siRNA knockdown in mouse embryonic stem cells.

Stephen Chen1, Andre Choo, Nai-Dy Wang

  • 1Stem Cell Group, Bioprocessing Technology Institute, 20 Biopolis Way, Singapore, Singapore.

Biotechnology Letters
|November 9, 2006
PubMed
Summary
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Optimizing small nucleic acid delivery into mouse embryonic stem cells (mESC) is crucial. Transfecting mESC 4 hours post-seeding significantly enhances delivery efficiency and gene silencing compared to overnight cultures.

Area of Science:

  • Biotechnology
  • Molecular Biology
  • Stem Cell Research

Background:

  • Efficient delivery of small nucleic acids into cells is essential for gene silencing applications.
  • Mouse embryonic stem cells (mESC) are notoriously difficult to transfect, posing challenges for experimental manipulation.
  • Optimizing transfection protocols is critical for successful gene knockdown studies in stem cells.

Purpose of the Study:

  • To evaluate the efficiency of small nucleic acid transfection in mouse embryonic stem cells (mESC).
  • To determine the optimal timing for transfection in mESC to achieve high delivery rates.
  • To establish a reliable method for assessing transfection efficiency using fluorescently labeled oligonucleotides.

Main Methods:

  • Utilized FAM-labeled oligo dT (FAMdT) as a fluorescent reporter to gauge transfection efficiency.

Related Experiment Videos

  • Compared transfection outcomes in overnight-grown mESC colonies versus mESC seeded 4 hours prior.
  • Quantified Oct-3/4 RNA transcript levels using quantitative real-time PCR to assess gene knockdown efficacy following siRNA delivery.

    • Main Results:

    • Transfection of overnight mESC colonies resulted in peripheral delivery and only a 40% decrease in Oct-3/4 RNA transcript levels.
    • Transfection of mESC 4 hours after seeding achieved over 90% successful cell transfection.
    • Quantitative real-time PCR confirmed approximately 90% Oct-3/4 RNA transcript knockdown in mESC transfected 4 hours post-seeding.

    Conclusions:

    • Timing of seeding significantly impacts small nucleic acid transfection efficiency in mESC.
    • A 4-hour post-seeding window provides optimal conditions for high-efficiency transfection and gene knockdown in mESC.
    • This optimized method offers an economical and efficient approach for transfecting difficult cell lines like mESC.