B6.129P2(Cg)-Rr27^tm4Msb/J

Stock number: 026687
Product name: H19^{Δ3.8kb-5'H19}
Research areas: Cell Biology Research, Developmental Biology Research, Research Tools

Description

The H19^{Δ3.8kb-5'H19} knockout allele has a 3.8 kbp deletion encompassing the entire differentially methylated domain (DMD or Rr27) and the 461 bp G-rich repeat element. Because this DMD coordinates the reciprocal expression of H19 and Igf2 by controlling access to shared enhancers, these mice may be useful in studying the role of this imprinting control region in the chromatin interactions and epigenetics of H19/Igf2 imprinting.

Detailed description

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.

This H19^{Δ3.8kb-5'H19} knockout allele has a 3.8 kbp deletion encompassing the entire DMD and the 461 bp G-rich repeat element. Mice homozygous for the H19^{Δ3.8kb-5'H19} knockout allele are viable and fertile, with tissue-specific disruption of imprinted expression of H19 and Igf2.

Development

The H19^{Δ3.8kb-5'H19} knockout allele was designed by Dr. Marisa S. Bartolomei (University of Pennsylvania) to have a 3.8 kbp deletion of chromosome 7 located ~2 kbp transcriptionally upstream of H19 and ~90 kbp transcriptionally downstream of Igf2. The deleted region includes the entire regulatory region 27 (Rr27; a differentially methylated domain [DMD]) and the nearby 461 bp G-rich repeat element.

The targeting vector was electroporated into 129P2/OlaHsd-derived E14.1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. Mice harboring the H19^{Δ3.8kb-5'H19neoR} allele were mated to Pou3f4-cre transgenic mice (on a C57BL/6J genetic background) to facilitate germline removal of a PGK-neo cassette. The resulting H19^{Δ3.8kb-5'H19} mice were subsequently backcrossed to C57BL/6J mice for at least ten generations (and the Pou3f4-cre transgene was removed) prior to sending black homozygous males to The Jackson Laboratory in 2012. 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

Symbol: Rr27^tm4Msb
Name: targeted mutation 4, Marisa S Bartolomei
MGI accession ID: MGI:3617971
Generation method: Targeted
Attributes: Null/Knockout; Modified regulatory region
Marker symbol: Rr27
Marker name: regulatory region 27
Chromosome: 7
Strain of origin: 129P2/OlaHsd
Molecular note: The targeting vector replaced 3.8 kb of sequence including the entire DMD and G-rich repeat element. Cre-mediated recombination removed the floxed neomycin resistance gene cassette.