Morpholino-mediated gene knockdown in mammalian organ culture.

We examined the role of semaphorin3a in ureteric bud (UB) branching morphogenesis using mouse metanephric organ culture [Tufro et al. (Mech Dev 125:558-568, 2008)]. In vitro UB injection of Sema3a antisense morpholino resulted in increased branching morphogenesis. Cellular and tissue uptake of oligonucleotides was facilitated by a peptide-mediated method. Our findings were validated by in vitro translation and in Sema3a null mice. This chapter describes a method to perfuse the UB lumen with fluorescein-labeled oligonucleotides bound to a peptide carrier.