A Diphtheria Toxin Negative Selection in RNA Interference Screening

RNA interference (RNAi) screening is a powerful technique for understanding the molecular biology of cancer and searching drug targets. Genes and their upstream activators that are essential for the survival of cancer cells often dictate cancer formation/progression. Hence, they are preferable therapeutic targets. Identifying these genes using RNAi is, however, problematic because knocking them down leads to cell death. Here we describe a diphtheria toxin (DT) negative selection method to circumvent the problem of cell death in RNAi screening. DT fails to kill mouse cells due to the lack of functional DT receptor (DTR). Thus, we first prepare a construct encoding a human functional DTR driven by the promoter of mouse Atf5, a gene essential for the survival of malignant glioma. Then a DT-sensitive mouse malignant glioma cell line is established by over-expressing this DTR. Finally, an RNAi screen is performed in this cell line and genes that activate Atf5 expression are identified. The negative selection approach described here allows RNAi screening to be used for identifying genes controlling cell survival in cancers or perhaps other human diseases with potential in therapeutic intervention.

Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news