RNA interference through the expression of small hairpin RNA (shRNA) molecules has become a very promising tool in reverse mouse genetics as it may allow inexpensive and rapid gene function analysis in vivo . However, the prerequisites for ubiquitous and reproducible shRNA expression are not well defined. Here we show that a single copy shRNA-transgene can mediate body-wide gene silencing in mice when inserted in a defined locus of the genome. The most commonly used promoters for shRNA expression, H1 and U6, showed a comparably broad activity in this configuration. Taken together, the results define a novel approach for efficient interference with expression of defined genes in vivo . Moreover, we provide a rapid strategy for the production of gene knockdown mice combining recombinase mediated cassette exchange and tetraploid blastocyst complementation approaches.