New approaches for modelling sporadic genetic disease in the mouse

Abstract

Sporadic diseases, which occur as single, scattered cases, are among the commonest causes of human morbidity and death. They result in a variety of diseases, including many cancers, premature aging, neurodegeneration and skeletal defects. They are often pathogenetically complex, involving a mosaic distribution of affected cells, and are difficult to model in the mouse. Faithful models of sporadic diseases require innovative forms of genetic manipulation to accurately recreate their initiation and pathogenesis. Such modelling is crucial to understanding these diseases and, by extension, to the development of therapeutic approaches to treat them. This article focuses on sporadic diseases with a genetic aetiology, the challenges they pose to biomedical researchers, and the different current and developing approaches used to model such disorders in the mouse.

Fig. 1.

Approaches to modelling somatic mutation in the mouse. (A) Conditional system. The gene encoding Cre recombinase is expressed from a tissue-specific promoter (TSP) and Cre catalyses the recombination between loxP sites (black arrowheads). As a result, the intervening sequence containing key exons (vertical black lines) is circularised and excised from the genome, so that it is lost later with cell division. (B) Tamoxifen-inducible system. A fusion gene that encodes Cre and a modified oestrogen ligand-binding domain (ER) is expressed from a TSP. The Cre-ER chimeric protein translocates to the nucleus in the presence of tamoxifen (Tam), resulting in recombination between loxP sites. (C) Tet-on/Tet-off inducible system. Tet-off: In the absence of doxycycline (Dox), the transcriptional activator tTA, which is expressed from a tissue-specific promoter, binds to the TRE to activate transcription of the downstream Cre transgene. If doxycycline is present, it binds to tTA preventing TRE binding and Cre transcription. Tet-on: In the presence of doxycycline, rtTA binds to TRE and activates Cre transcription. This binding does not happen in the absence of doxycycline. (D) Transposition. Sleeping Beauty (SB) or PiggyBac (PB) transposase expressed from a constitutive promoter (CP) or a TSP mobilises a transposon (T) from a chromosomal donor site to a distant site on another chromosome where it can disrupt a gene (insertional mutagenesis). (E) Zinc finger nuclease (ZFN) system. ZFN cDNA is delivered by a retrovirus and expressed from a CP. The ZFN protein generates a targeted double-strand break, which is then repaired by the homologous recombination machinery (HRM) of the cell using a co-delivered extra-chromosomal DNA sequence (ECD) that carries a mutant or reverted exon (*). The mutation or reversion is thus incorporated into the cellular genome. (F) Mosaic analysis with double markers (MADM). Two reciprocally chimeric marker genes (GFP-RFP and RFP-GFP) are targeted to the same locus on homologous chromosomes. One is associated with a wild-type allele (+, GFP-RFP in this example) and one with a knockout allele (–, RFP-GFP in this example). In cells that are heterozygous for the two chimeric markers, Cre mediates recombination between non-sister chromatids at loxP sites during the G2 phase of the cell cycle. When this is followed by X segregation (where sister chromatids segregate into two different daughter cells), it generates singly labelled cells (green or red) with an altered genotype (−/− or +/+). When G2 recombination is followed by Z segregation (where sister chromatids segregate into the same daughter cell), or after G0 or G1 recombination, either colourless or double-coloured cells are generated whose genotype is unaltered (+/–). GFP, green fluorescent protein; RFP, red fluorescent protein.