Berlivet Soizik, Hmitou Isabelle, Picaud Hélène, Gérard Matthieu
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette, France.
Methods Mol Biol. 2017;1622:91-100. doi: 10.1007/978-1-4939-7108-4_7.
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.
CRISPR/Cas9技术的发展为靶向基因改变提供了强大的方法。然而，研究细胞存活所必需的基因的功能仍然存在问题，因为这些基因的基因消融会导致细胞死亡。因此，无法获得在靶向基因处重组且基因产物完全耗尽的细胞。RNA干扰非常适合研究必需基因，但这种方法通常会导致靶向基因产物的部分耗尽，这可能会导致误解。我们之前开发了pHYPER shRNA载体，这是一种基于包含H1基因的2.5kb小鼠基因组片段的高效RNA干扰载体。我们在此提供一种基于pHYPER的方案，该方案经过优化，用于研究小鼠胚胎干细胞中必需基因产物的功能。
