FACS-Based High-Throughput Functional Screening of Genetic Libraries for Drug Target Discovery

Abstract

A primary aim of functional genomics in pharmaceutical applications is to identify genes whose function is critical to maintaining a disease state and to determining whether therapeutic modulation of this function results in a beneficial clinical response. However, although many genomic approaches can identify disease-associated genes, lengthy follow-up studies are usually required to determine which genes are functionally important and are causally linked to a given disease. In contrast, retrovirally mediated functional genetic screening approaches enable rapid identification of physiologically relevant targets. Genetic screens are designed to detect functional changes that result in changes in cellular function that correlate with disease amelioration. Retroviruses possess unique properties that allow delivery of complex libraries of potential genetic effectors to a variety of cell types. These effectors can perturb specific interactions required to achieve a complete functional response and establish a direct relationship with a cellular function. Functional screens are employed to select for cells endowed with a desired genetic effector–induced change in phenotype. Identification of a genetic effector that causes an altered cellular phenotype that correlates with clinical benefit can explicate critical signaling components suitable for therapeutic intervention. Flow cytometry represents a uniquely powerful methodology to monitor complex multiparametric changes of individual cells in large populations. In conjunction with recent advances in retroviral expression systems, the sensitivity and speed of flow cytometry enables a highly efficient functional screening of complex libraries in a wide range of cell-based assays. In this chapter, we discuss the process of functional genetic screening and show specific examples of its implementation. We focus particularly on the critical parameters involved in the design and execution of functional genetic screening approaches based on FACS (fluorescence activated cell sorter). Retroviruses provide a powerful method of introducing genes into mammalian cells in an efficient and stable manner. Recent advances in retroviral vector technology and packaging systems have extended their application to allow efficient and stable delivery of highly diverse libraries encoding various types of genetic effectors, including cDNAs, peptides, and ribozymes, into a broad range of cell types.