A cre-inducible DUX4 transgenic mouse model for investigating facioscapulohumeral muscular dystrophy

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.

Transgenic mice expressing tunable levels of DUX4 develop characteristic facioscapulohumeral muscular dystrophy-like pathophysiology ranging in severity

“…The ACTA1-MCM Cre driver line refers to B6.Cg-Tg(ACTA1-cre/Esr1*)2Kesr/J (JAX 025750) [48] and Rosa26 NZG refers to FVB.Cg-Gt(ROSA)26Sor<tm1(CAG-lacZ,-EGFP)Glh>/J (JAX 012429) [49], both purchased from Jackson Labs (Bangor, Maine). FLExDUX4 (FLExD) mice were generated and characterized by the PL Jones lab [41] and are available from Jackson Labs (JAX 028710). Mice were genotyped as described [41].…”

“…FLExDUX4 (FLExD) mice were generated and characterized by the PL Jones lab [41] and are available from Jackson Labs (JAX 028710). Mice were genotyped as described [41].…”

“…Total RNA was extracted from dissected mouse muscles homogenized in 10 volumes of TRIzol (ThermoFisher) using the TissueLyser LT (Qiagen), as per manufacturer's instructions followed by on-column DNaseI treatment and clean-up using the RNeasy mini kit (Qiagen). Quantitative DUX4-fl mRNA expression was analyzed using nested qRT-PCR, as described [41,50].…”

“…Expression of DUX4 target genes was analyzed by qPCR using 5 to 10ng cDNA, as described [30,41]. All oligonucleotide primer sequences for DUX4-fl, downstream targets, and 18S rRNA are previously reported [29].…”

“…We have previously reported the generation of the FLExDUX4 (FLExD) conditional DUX4-fl transgenic line of mice, which contains a DUX4 transgene engineered into the Rosa26 locus using the FLEx directional switch system [39,40] to bypass the embryonic lethality from leaky embryonic expression of this transgene [41]. Upon Cre-mediated induction, the transgene recombines to express DUX4-fl under control of the Rosa26 gene promoter ( Figure 1A).…”