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A diphtheria toxin negative selection in RNA interference screening
Zhi Sheng1, Susan F Murphy, Sujuan Guo
1Virginia Tech Carilion Research Institute, 2 Riverside Circle, Roanoke, VA, 24016, USA, zhisheng@vtc.vt.edu.
Abstract:
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.
Insights
This study introduces a novel diphtheria toxin negative selection method for RNA interference (RNAi) screening. This approach overcomes cell death issues, enabling the identification of essential cancer survival genes and potential therapeutic targets.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- RNA interference (RNAi) screening is vital for cancer research and drug target discovery.
- Identifying essential survival genes via RNAi is challenging due to cell death upon knockdown.
- Targeting genes crucial for cancer progression is a key therapeutic strategy.
Purpose of the Study:
- To develop a negative selection method for RNAi screening that circumvents cell death.
- To identify genes essential for cancer cell survival and potential therapeutic targets.
- To enable RNAi screening for genes controlling cell survival in malignant glioma.
Main Methods:
- A diphtheria toxin (DT) negative selection strategy was employed.
- A construct for human diphtheria toxin receptor (DTR) driven by the mouse Atf5 promoter was created.
- A DT-sensitive mouse malignant glioma cell line overexpressing DTR was established for RNAi screening.
Main Results:
- The developed method successfully enabled RNAi screening in DT-sensitive cells.
- Genes that activate Atf5 expression, crucial for glioma survival, were identified.
- The negative selection approach proved effective in identifying genes controlling cell survival.
Conclusions:
- The diphtheria toxin negative selection method facilitates RNAi screening for essential cancer survival genes.
- This technique offers a powerful approach for discovering therapeutic targets in cancer and other diseases.
- The method overcomes limitations of traditional RNAi screening, enabling broader applications in disease research.
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