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Updated: Apr 27, 2026
In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing
Published on: May 5, 2023
Flow cytometry-based functional selection of RNA interference triggers for efficient epi-allelic analysis of
David R Micklem, Magnus Blø, Petra Bergström
1Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway. jim.lorens@biomed.uib.no.
Background:
The dose-response relationship is a fundamental pharmacological parameter necessary to determine therapeutic thresholds. Epi-allelic hypomorphic analysis using RNA interference (RNAi) can similarly correlate target gene dosage with cellular phenotypes. This however requires a set of RNAi triggers empirically determined to attenuate target gene expression to different levels.
Results:
In order to improve our ability to incorporate epi-allelic analysis into target validation studies, we developed a novel flow cytometry-based functional screening approach (CellSelectRNAi) to achieve unbiased selection of shRNAs from high-coverage libraries that knockdown target gene expression to predetermined levels. Employing a Gaussian probability model we calculated that knockdown efficiency is inferred from shRNA sequence frequency profiles derived from sorted hypomorphic cell populations. We used this approach to generate a hypomorphic epi-allelic cell series of shRNAs to reveal a functional threshold for the tumor suppressor p53 in normal and transformed cells.
Conclusion:
The unbiased CellSelectRNAi flow cytometry-based functional screening approach readily provides an epi-allelic series of shRNAs for graded reduction of target gene expression and improved phenotypic validation.
Insights
Researchers developed CellSelectRNAi, a flow cytometry method for selecting RNA interference (RNAi) triggers that precisely reduce gene expression. This enables accurate gene dosage studies and identifies functional thresholds, like for the p53 tumor suppressor.
Area of Science:
- Pharmacology and Molecular Biology
- Functional Genomics
- Cancer Research
Background:
- The dose-response relationship is crucial for determining therapeutic thresholds.
- Epi-allelic hypomorphic analysis using RNA interference (RNAi) links gene dosage to cellular phenotypes.
- This analysis requires RNAi triggers that attenuate gene expression to specific levels.
Purpose of the Study:
- To develop a method for unbiased selection of RNAi triggers for precise gene knockdown.
- To enable robust epi-allelic analysis in target validation studies.
- To establish a functional threshold for the tumor suppressor p53.
Main Methods:
- Developed CellSelectRNAi, a flow cytometry-based functional screening approach.
- Used a Gaussian probability model to infer knockdown efficiency from shRNA frequency profiles.
- Generated a hypomorphic epi-allelic cell series of shRNAs.
Main Results:
- CellSelectRNAi enables unbiased selection of shRNAs for predetermined gene knockdown levels.
- Knockdown efficiency is accurately inferred from shRNA sequence frequency profiles.
- A functional threshold for p53 was revealed in normal and transformed cells.
Conclusions:
- CellSelectRNAi provides an unbiased method for generating epi-allelic shRNA series.
- This approach allows for graded reduction of target gene expression.
- Facilitates improved phenotypic validation in gene function studies.
