Enrichment during transdominant genetic experiments using a flow sorter

Abstract

Background: Flow cytometry, in combination with retroviral expression libraries, is a powerful tool for genetic experimentation in mammalian cells. Expression libraries are transduced into cells engineered with a fluorescent reporter. Sorting for either bright or dim cells allows enrichment for specific inhibitors that alter reporter activity. This strategy has been used to isolate peptides and RNAs that either activate or suppress defined biochemical pathways. Methods: Several variables contribute to the enrichment process: (1) the background of the fluorescence bioassay; (2) the mean fluorescence ratio between the induced and noninduced reporter cell populations; (3) the genetic penetrance, or strength, of the inhibitor; and (4) the multiplicity of infection (MOI). An experimental and theoretical analysis, including computer modeling, of these issues in the context of a mammalian cell bioassay was undertaken. Results: MOI measurements were shown to be problematic. High MOI had little effect on enrichment early in the cycling process but a significant effect at later stages. Penetrance and background were critical throughout the process. Enrichments within about twofold of the theoretical maximum were observed. Conclusions: Caution should be exercised in MOI determination because of the danger of significant underestimation. High MOI is potentially advantageous early in the selection process but hinders enrichment in the later rounds. Modeling shows that MOI, assay background and clone penetrance are the principal variables that determine the success of transdominant selections by FACS. © 2001 Wiley-Liss, Inc.