Abstract

The Double homeobox 4 (DUX4) gene is an important regulator of early human development and its aberrant expression is causal for facioscapulohumeral muscular dystrophy (FSHD). The DUX4-full length (DUX4-fl) mRNA splice isoform encodes a transcriptional activator; however, DUX4 and its unique DNA binding preferences are specific to old-world primates. Regardless, the somatic cytotoxicity caused by DUX4 expression is conserved when expressed in cells and animals ranging from fly to mouse. Thus, viable animal models based on DUX4-fl expression have been difficult to generate due in large part to overt developmental toxicity of low DUX4-fl expression from leaky transgenes. We have overcome this obstacle and here we report the generation and initial characterization of a line of conditional floxed DUX4-fl transgenic mice, FLExDUX4, that is viable and fertile. In the absence of cre, these mice express a very low level of DUX4-fl mRNA from the transgene, resulting in mild phenotypes. However, when crossed with appropriate cre-driver lines of mice, the double transgenic offspring readily express DUX4-fl mRNA, protein, and target genes with the spatiotemporal pattern of nuclear cre expression dictated by the chosen system. When cre is expressed from the ACTA1 skeletal muscle-specific promoter, the double transgenic animals exhibit a developmental myopathy. When crossed with tamoxifen-inducible cre lines, DUX4-mediated pathology can be induced in adult animals. Thus, the appearance and progression of pathology can be controlled to provide readily screenable phenotypes useful for assessing therapeutic approaches targeting DUX4-fl mRNA and protein. Overall, the FLExDUX4 line of mice is quite versatile and will allow new investigations into mechanisms of DUX4-mediated pathophysiology as well as much-needed pre-clinical testing of DUX4-targeted FSHD interventions in vivo.

Highlights

  • These transcripts encode the nonpathogenic Double homeobox 4 (DUX4)-short (DUX4-S) isoforms, which have a dominant negative effect on DUX4-FL; it was important to prevent their expression from our DUX4 transgene [5]

  • Following transfection of our DUX4 expression construct into murine C2C12 cells, we confirmed its functionality by RT-PCR, and the lack of detectable expression of DUX4-s mRNA (S1 Fig)

  • The transgene was flanked by heterologous loxP sites that recombine unidirectionally upon exposure to Cre recombinase, resulting in DUX4-full length (DUX4-fl) mRNA expression under transcriptional control of the Rosa26 promoter

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Summary

Objectives

Our goal was to generate a viable and fertile transgenic mouse model containing an intact human DUX4 transgene that, upon controlled induction, produces mosaic expression and a myopathic phenotype [4, 10, 18]

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