Efficient Depletion of Essential Gene Products for Loss-of-Function Studies in Embryonic Stem Cells
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 wordsRNA interference shRNA Embryonic stem cell pHYPER Essential genes Electroporation Puromycin selection
- 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.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