Genetic ablation of BMPR2 function in mice. (A) Ubiquitous expression of shRNA transgene at a defined site in the mouse genome. A modified Rosa26 locus was generated to allow Cre-mediated RMCE through the heterotypic loxP and lox511 sequences inserted into the locus by gene targeting. Next, homologous recombination in ES cells between the lox sites in the exchange vector and the modified Rosa26 RMCE locus led to integration of the U6-shRNA expression cassette. (B) In vivo RNA interference. Bmpr2 mRNA expression in various tissues from wild-type (WT) littermates or Bmpr2 knockdown (KD) mice was assessed by real-time quantitative PCR. Values are means plus or minus SD. (C) Knockdown of BMPR2 protein expression. BMPR2 was detected in immunoblots of tissue extracts. β-actin and α-tubulin in the same blots serve as internal control.