Efficient in vivo manipulation of alternative pre-mRNA splicing events using antisense morpholinos in mice

Mammalian pre-mRNA alternative splicing mechanisms are typically studied using artificial minigenes in cultured cells, conditions that may not accurately reflect the physiological context of either the pre-mRNA or the splicing machinery. Here we describe a strategy to investigate splicing of normal endogenous full-length pre-mRNAs under physiological conditions in live mice. This approach employs antisense vivo-morpholinos to mask cis-regulatory sequences, or to disrupt splicing factor expression, allowing functional evaluation of splicing regulation in vivo. We applied this strategy to gain mechanistic insight into alternative splicing events involving exons 2 and 16 (E2 and E16) that control structure and function of cytoskeletal protein 4.1R. In several mouse tissues, inclusion of E16 was substantially inhibited by interfering with a splicing enhancer mechanism, using a target-protector morpholino that blocked Fox2-dependent splicing enhancers in intron 16, or a splice-blocking morpholino that disrupted Fox2 expression directly. For E2, alternative 3′ splice site choice is coordinated with upstream promoter use, across a long 5′ intron, such that exon 1A splices almost exclusively to the distal acceptor (E2dis). Vivo-morpholinos were used to test the in vivo relevance of a deep intron element previously proposed to determine use of E2dis via a two-step intrasplicing model. Two independent vivo-morpholinos designed against this intronic regulatory element abrogated intrasplicing, robustly switching exon 1A splicing to the proximal acceptor (E2prox). This finding strongly supports the in vivo physiological relevance of intrasplicing. Vivo-morpholinos represent a powerful tool for alternative splicing studies in vivo, and may facilitate exploration of alternative splicing networks in vivo.