UCSF

Transgenic methods to manipulate CD4 T lymphocytes in vivo via forced expression of TCR transgenes and targeted "knockout" of individual genes by Cre-lox technology are fundamental to modern immunology. However, efforts to scale up functional analysis by modifying expression of larger numbers of genes in T cells ex vivo have proven surprisingly difficult. Early RNA interference experiments achieved successful small RNA transfection by using very high concentrations of short-interfering RNA (siRNA) [1], but primary T cells are generally resistant to standard electroporation, cationic liposome-, and calcium phosphate-mediated transfection methods. Moreover, although viral vectors can successfully introduce DNA fragments of varying length, expression of these constructs in primary T cells is low efficiency and the subcloning process laborious. In this context, the relatively recent discovery of dozens of highly expressed microRNAs (miRNAs) in the immune system provides both an opportunity and a new challenge [2, 3]. How can we query the miRNAome of a cell to assign particular roles to individual miRNAs? Here, we describe an optimized technique for efficient and reproducible transfection of primary mouse CD4 T cells in vitro with synthetic miRNA mimics.