The impact of alternative splicing in vivo: mouse models show the way

Abstract

Alternative splicing is widely believed to have a major impact on almost all biological processes since it increases proteome complexity and thereby controls protein function. Recently, gene targeting in mice has been used to create in vivo models to study the regulation and consequences of alternative splicing. The evidence accumulated so far argues for a nonredundant, highly specific role of individual splicing factors in mammalian development, and furthermore, demonstrates the importance of distinct protein isoforms in vivo. In this review, we will compare phenotypes of mouse models for alternative splicing to crystallize common themes and to put them into perspective with the available in vitro data.

FIGURE 1.

Strategies that have been used to create splicing factor or isoform-specific knockout mice. Three exons (A–C) of the gene of interest are schematically shown as filled boxes (blue). (a) Large-scale random retroviral insertion of marker genes in the genome of murine ES cells, and subsequent sequencing of the integration site has been used to create libraries of targeted ES cells that are commercially available. Using this approach, stop codons, loxP sites, and other alterations can be introduced along with the marker gene into the gene of interest. (b) In a classical knockout approach, the whole gene, or a part thereof, is replaced by a selectable marker, e.g., a neomycin-resistance gene. To avoid potential effects of the neo-cassette, a version flanked by loxP sites can be used that can be removed by expression of the site-specific Cre recombinase. If only parts of the gene of interest are removed, as shown here, the expression of a truncated protein should be taken into consideration. (c) In a conditional knockout approach, the gene of interest is replaced by a version that carries flanking loxP sites (floxed) that can be removed by tissue-specific expression of Cre recombinase. Introduction of the floxed allele also requires the presence of a (removable) selectable marker (data not shown). (d) Knockin mutants are used to replace parts of the gene of interest with an altered version. For example, a stop codon can be introduced to abrogate expression of all isoforms carrying exon B, but allowing expression of A-C isoforms. A similar technique, also resulting in a depletion of isoforms containing exon B, is the destruction of the splice sites of the respective exon. Finally, one exon can be replaced by another exon or an unrelated coding region, e.g., to obtain the expression of an isoform in a tissue, where it is usually not expressed. This technique requires careful consideration of the targeting construct, as exonic regulatory sequences of the deleted exon might be important for tissue-specific splicing of the gene.