Chapter Eight - Rapid Creation of Stable Mammalian Cell Lines for Regulated Expression of Proteins Using the Gateway® Recombination Cloning Technology and Flp-In T-REx® Lines

Abstract

The biochemical analysis of cellular processes in mammalian cells is often facilitated by the creation of cell lines coexpressing or overexpressing an affinity-tagged wild-type or mutant protein of interest in an inducible or noninducible stable manner (Malik and Roeder, 2003). The affinity tag allows for standardization of purification protocols to characterize interacting proteins or nucleic acids and minimizes the need for generating protein-specific antibodies at the early stages of analysis (for more information on affinity tags, see Purification of His-tagged proteins, Affinity purification of a recombinant protein expressed as a fusion with the maltose-binding protein (MBP) tag, Purification of GST-tagged proteins, Protein Affinity Purification using Intein/Chitin Binding Protein Tags, Immunoaffinity purification of proteins or Strep-tagged protein purification). The establishment of stable cell lines with inducible expression is critical to studying proteins that reduce cell growth and/or viability upon overexpression. Over the past several years, our laboratory has developed an expression platform for analyzing RNA-interacting proteins, including the establishment of stable mammalian cell lines expressing proteins of interest using a recombination-based cloning technology (Landthaler et al., 2008; Hafner et al., 2010). Our aim is to determine the mRNA targets of the hundreds of RNA-binding proteins encoded in the human genome by the isolation and molecular characterization of their ribonucleoprotein complexes (RNPs).