Promoter-based expression of short hairpin RNAs (shRNAs) may in principle provide stable silencing of genes in any tissue. As for all approaches that require transgene expression, safe delivery is the biggest obstacle, but toxicity can also occur via expression of the sequence itself. Innate immunity mechanisms can be triggered by expressed hairpin RNAs, critical cellular factors can be saturated, and genes other than the intended target can be silenced. Nevertheless, shRNAs constitute a valuable tool for in vivo research and have great therapeutic potential if the challenges with delivery and side effects are appropriately addressed.