MicroRNA Activity in B Lymphocytes

Gene expression regulation by miRNAs has been reported to control key aspects of B cell differentiation and function (Chen et al., Science 303:83–86, 2004; Xiao et al., Cell 131:146–159, 2007; O’Carroll et al., Genes Dev. 21:1999–2004, 2007; Koralov et al. Cell 132:860–874, 2008; Rodriguez et al., Science 316:608–611, 2007; Costinean et al., Proc Natl Acad Sci USA 103:7024–7029, 2006; Thai et al., Science 316:604–608, 2007; Vigorito et al., Immunity 27:847–859, 2007; Dorsett et al., Immunity 28:630–638, 2008; Teng et al., Immunity 28:621–629, 2008; de Yebenes et al., J Exp Med 205:2199–2206, 2008; He et al., Nature 435:828–833, 2005; Ventura et al. Cell 132:875–886, 2008; Xiao et al., Nat Immunol 9:405–414, 2008). In this chapter, we describe the methodology used to perform a functional screening of a miRNA library to identify miRNAs relevant for mature B cell function in primary mouse B cells. These procedures include the construction of a miRNA library and the expression of individual miRNA clones in spleen B cells, as well as the description of functional assays used to determine the impact of miRNA expression on several aspects of B cell function, such as proliferation, apoptosis, and class switch recombination.