Chapter title |
Efficient Depletion of Essential Gene Products for Loss-of-Function Studies in Embryonic Stem Cells
|
---|---|
Chapter number | 7 |
Book title |
RNAi and Small Regulatory RNAs in Stem Cells
|
Published in |
Methods in molecular biology, July 2017
|
DOI | 10.1007/978-1-4939-7108-4_7 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7106-0, 978-1-4939-7108-4
|
Authors |
Soizik Berlivet, Isabelle Hmitou, Hélène Picaud, Matthieu Gérard, Berlivet, Soizik, Hmitou, Isabelle, Picaud, Hélène, Gérard, Matthieu |
Editors |
Baohong Zhang |
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. |
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