RNAi and Small Regulatory RNAs in Stem Cells pp 91-100

Part of the Methods in Molecular Biology book series (MIMB, volume 1622) | Cite as

Efficient Depletion of Essential Gene Products for Loss-of-Function Studies in Embryonic Stem Cells

  • Soizik Berlivet
  • Isabelle Hmitou
  • Hélène Picaud
  • Matthieu Gérard
Protocol

Abstract

The development of the CRISPR/Cas9 technology has provided powerful methods to target genetic alterations. However, investigating the function of genes essential for cell survival remains problematic, because genetic ablation of these genes results in cell death. As a consequence, cells recombined at the targeted gene and fully depleted of the gene product cannot be obtained. RNA interference is well suited for the study of essential genes, but this approach often results in a partial depletion of the targeted gene product, which can lead to misinterpretations. We previously developed the pHYPER shRNA vector, a high efficiency RNA interference vector, which is based on a 2.5-kb mouse genomic fragment encompassing the H1 gene. We provide here a pHYPER-based protocol optimized to study the function of essential gene products in mouse embryonic stem cells.

Key words

RNA interference shRNA Embryonic stem cell pHYPER Essential genes Electroporation Puromycin selection 

References

  1. 1.
    Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498CrossRefPubMedGoogle Scholar
  2. 2.
    Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16:948–958CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550–553CrossRefPubMedGoogle Scholar
  4. 4.
    Paul CP, Good PD, Winer I, Engelke DR (2002) Effective expression of small interfering RNA in human cells. Nat Biotechnol 20:505–508CrossRefPubMedGoogle Scholar
  5. 5.
    Sui G, Soohoo C, Affar el B, Gay F, Shi Y, Forrester WC (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci U S A 99:5515–5520CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Miyagishi M, Taira K (2002) U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol 20:497–500CrossRefPubMedGoogle Scholar
  7. 7.
    Lee NS, Dohjima T, Bauer G et al (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 20:500–505Google Scholar
  8. 8.
    Houlard M, Berlivet S, Probst AV et al (2006) CAF-1 is essential for heterochromatin organization in pluripotent embryonic cells. PLoS Genet 2:e181Google Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Soizik Berlivet
    • 1
  • Isabelle Hmitou
    • 1
  • Hélène Picaud
    • 1
  • Matthieu Gérard
    • 1
  1. 1.Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-SudUniversité Paris-SaclayGif-sur-YvetteFrance

Personalised recommendations