Fig. 1

DUX4 poly A signal read through and the effect of removing the SV40 pA signal. a Schematic of transgene integration in iDUX4[2.7] mice and primers/probe used for detection of transcript proximal to and distal of the DUX4 pA signal. Note the SV40 poly A signal downstream of the DUX4 poly A signal. b RTqPCR with reverse primers on either side of the DUX4 pA signal in PDGFRα⁺ integrin α7^– (fibroadiopgenic) and PDGFRα^– integrin α7⁺ (myogenic) sorted cultured primary muscle cells. n = 3 technical replicates. Note the large amount of transcript that is detected by the primer downstream of the DUX4 pA, indicating that the DUX4 poly A is not efficient and allows a high level of read through. In the iDUX[2.7] mouse, these read through transcripts are stabilized by the SV40 poly A, potentiating higher basal levels of DUX4 in the off state. c The transgene is targeted 5ʹ of HPRT, is regulated by a second generation tetracycline-response element, includes DUX4 and 3ʹ UTR including the DUX4 poly A signal. d Mice were made in which the SV40 pA was removed, leaving only the endogenous, less efficient, DUX4 pA. Viability is now near-normal for male carriers. Summary of 174 genotypes of 3-week old mice from 21 litters of iDUX4pA females backcrossed to males with a WT chrX. Results are not significantly different from expected for the hypothesis of no selection against iDUX4pA ( χ², two tailed p = 0.2956), but are strongly diverged from expected (p < 0.0001) for the hypothesis that iDUX4pA is selected against at the same level as observed against iDUX4[2.7]²⁰. Thus, we conclude that the presence of the DUX4 poly A makes the transgene less toxic, with viability to 3 weeks not different from WT in the absence of dox