The H19lxDMD floxed allele allows cre-mediated deletion of a 1.6 kbp region of the differentially methylated domain (DMD or Rr27). This DMD coordinates the reciprocal expression of H19 and Igf2 by controlling access to shared enhancers. These mice may be used to generate temporal and tissue-specific deletions of this imprinting control region for studying the chromatin interactions and epigenetics involved in H19/Igf2 imprinting.
Marisa S Bartolomei, University of Pennsylvania
Imprinted expression of the H19 and Igf2 loci are dependent upon a differentially methylated domain (DMD or Rr27) that coordinates the reciprocal maternal/paternal expression of H19 and Igf2 by controlling access to shared enhancers. Specifically, on the maternal allele, the DMD acts as a methylation-sensitive insulator that results in H19 expression and Igf2 inactivation. The four CpG-rich regions in the DMD bind CCCTC-binding factor (CTCF) on the hypomethylated maternal allele, subsequently blocking the Igf2 gene from the enhancers shared by H19 and Igf2. On the paternal allele, the hypermethylation of DMD concomitantly methylates H19, resulting in H19 inactivation and Igf2 expression.
The H19lxDMD floxed allele has loxP sites flanking a 1.6 kbp KpnI-HindIII fragment of DMD, including three of the four CpG-rich regions. Mice homozygous for the H19lxDMD floxed allele are viable and fertile. After Cre recombinase-deletion of the floxed 1.6 kbp DMD region, the resulting ΔDMD mice exhibit tissue-specific disruption of imprinted expression of H19 and Igf2.
The H19lxDMD floxed allele was designed by Dr. Marisa S. Bartolomei (University of Pennsylvania) to have loxP sites flanking the 1.6 kbp KpnI-HindIII fragment of the regulatory region 27 (Rr27; a differentially methylated domain [DMD] located ~2 kbp transcriptionally upstream of H19 and ~90 kbp transcriptionally downstream of Igf2).
The targeting vector was electroporated into 129P2/OlaHsd-derived E14.1 embryonic stem (ES) cells. Correctly targeted ES cells (with the H19lxDMDneo genotype) were then transiently transfected with a Cre recombinase-expressing plasmid. The resulting ES cells with the H19lxDMD genotype (retaining the floxed DMD with the PGK-neo deleted) were injected into recipient blastocysts. Chimeric mice were bred with C57BL/6J mice to establish the H19lxDMD colony. The donating investigator reports that H19lxDMD mice were subsequently backcrossed to C57BL/6J mice for a total of at least four generations prior to sending black homozygous males to The Jackson Laboratory in 2011. Upon arrival, males were used to cryopreserve sperm. To establish the living mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred mice (Stock No. 000664).
|Allele Name||targeted mutation 2, Marisa S Bartolemei|
|Allele Type||Targeted (Conditional ready (e.g. floxed), No functional change, Modified regulatory region)|
|Allele Synonym(s)||H19lxDMD; H19tm2Msb|
|Gene Symbol and Name||Rr27, regulatory region 27|
|Strain of Origin||129P2/OlaHsd|
|Molecular Note||A targeting vector was designed to insert loxP sites around the 1.6 DMD sequence. A floxed neo was removed via cre mediated recombination.|
When maintaining a live colony, homozygous mice may be bred together.
When using the H19lxDMD mouse strain in a publication, please cite the originating article(s) and include JAX stock #026688 in your Materials and Methods section.