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


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 


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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

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