Abstract LB-421: Dual shRNA technique to screen gene-to-gene interaction

Abstract

Abstract Background: Synthetic lethality screening in human cells is one of the most powerful tools to identify potential therapeutic targets in a genotype specific manner and this screening has been performed in isogenic models of matched cell-line pairs. The procedure of establishing and isolating isogenic cell lines can be arduous. And despite this laborious work, specific target(s) identified in this specific isogenic cell line may not be valid in other similar cells and experimental conditions. Therefore, we have optimized a fast and robust dual shRNA technique to screen gene-to-gene interaction. Methods and Results: To create a matched cell line pair, we utilized a bicistronic vector containing the transmembrane and extracellular domains of the mouse T-cell surface glycoprotein CD8 alpha, co-expressed with a specific shRNA, to model a specific gene deficiency. This retroviral vector was transduced into target cells, and the positively transduced cells were isolated at two days post-infection using MACS separation columns with mouse CD8a-specific microbeads. This yielded greater than 95% purity. At this time point, the decreased expression of specific gene by shRNA was minimal. After recovery from beads purification, the second bicistronic shRNA vector was introduced. This second vector contained a puromycin selectable marker with GFP and an shRNA targeting the proposed synthetic lethal gene. After second transduction, cells were selected under puromycin allowing two doubling times. The purity was 96-98% based on GFP signal measured by FACS at the end of puromycin selection. The dual-gene knockdown by sequentially introduced shRNAs achieved 70-90% depletion of each gene product. This level of knockdown was maintained for up to three weeks without significant loss of purity. After maintaining the cells in culture for several weeks CD8a purity did decrease to 70-80%, but was restored to 98-99% purity after re-applying the separation columns. The second level of purity was maintained even longer than single purification. Conclusion: In summary, we have developed a fast and efficient dual shRNA technique to apply to shRNA experimentation, obviating the need for establishing conventional isogenic cell line models. Due to its fast and simple procedure, this can be simultaneously performed in multiple cell lines. This technique can easily be adapted to screen shRNA libraries for the discovery of unknown synthetic lethal interactions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-421. doi:1538-7445.AM2012-LB-421