Endonuclease-mediated deletion of the macrosatellite repeat Dxz4, which includes the lncRNA gene 4933407K13Rik.

Adopted from PMID:31738164: During the process of X chromosome inactivation (XCI) the inactive X chromosome forms a compact chromatin structure known as the ‘Barr body’. Recent studies using chromosome conformation capture-based methods have identified that the Barr body folds into two megadomains and forms a network of long-range interactions termed superloops that are directed by the non-coding X-linked loci Firre and Dxz4. Moreover, the Firre locus is transcribed into the long non-coding RNA (lncRNA) Firre that plays a role in nuclear organization. Studies in mouse cell lines found that the deletion of these loci has minimal impact on X chromosome biology beyond the loss of these chromatin structures, though the phenotypic consequences of their deletion throughout mammalian development have not been addressed. In order to test the in vivo role of Firre and Dxz4 loci both individually and in combination, the donor generated three knockout mouse strains: two carrying a single locus deletion of either Firre or Dxz4 and one carrying a double locus deletion of Firre in conjunction with Dxz4 (MMRRC:66756). They found that mice carrying a single or double deletion of Firre and Dxz4 were viable, fertile, and showed no defect in random or imprinted XCI. However, the lack of these loci results in many dysregulated genes on autosomes in an organ-specific manner (most of them in the spleen). The largest transcriptional effect on autosomes was Firre locus-dependent, suggesting a role for the Firre RNA in autosomal gene regulation. However, the donor also observed Dxz4-dependent transcriptional effect on autosomes, suggesting that structural changes of the Barr body may lead to autosomal gene dysregulation.

The Dxz4 single deletion strain (ChrX:75,721,164–75,764,733 mm10) was generated by co-injecting Cas9 mRNA (200 ng/µl) together with two guide RNA’s that span the Dxz4 locus (50 ng/µl each) into pronuclear stage 3 (PN3) zygotes isolated after mating superovulated B6D2F1 female mice (JAX Strain #100006) with C57BL/6 males as previously described (PMID:23643243). The strain was backcrossed twice to C57BL/6J, followed by 10 sib-matings.

The MMRRC Centers have developed a genetic QC pipeline using MiniMUGA array genotyping to provide additional information on strain backgrounds for MMRRC congenic and inbred strains. For more information on when data may be available, or to request genotyping for a strain of interest, please contact mmrrc@ucdavis.edu. Older strains may not have this information.

John Rinn, Ph.D., University of Colorado Boulder.