B6.129S-Dmtn^{tm1c(KOMP)Mbp}/AhcJ

Stock number: 034593
Product name: Epb4.9^fl
Research areas: Hematological Research, Research Tools

Description

Epb4.9^fl/fl mice have loxP sites flanking exons 5-8 of the dematin actin binding protein (Dmnt) gene. These mice are useful for studying membrane cytoskeleton stability.

Detailed description

Epb4.9^fl/fl mice have loxP sites flanking exons 5-8 of the dematin actin binding protein (Dmnt) gene. Dmnt encodes Epb4.9, an actin binding and bundling protein associated with the spectrin-actin junctions of mature erythrocytes. The primary structure of dematin includes a loosely folded core domain and a compact headpiece domain. Headpiece domain is essential for the actin-bundling activity of dematin, and phosphorylation of the headpiece domain by cAMP-dependent protein kinase negatively regulates its actin bundling activity in vitro. Mice that are homozygous for this Epb4.9^fl/fl allele are viable and fertile. When bred to mice that express tissue-specific Cre recombinase, resulting offspring will have exons 5-8 deleted in the Cre-expressing tissues.



When bred to a Cre recombinase-expressing strain, removal of exons 5-8 introduces a frame shift and premature stop codon in exon 10, triggering nonsense-mediated decay of the mutant transcript. Homozygous full-length knock-out (FLKO) mice are viable and fertile, but are paler than wildtype (WT) pups at birth. At 3 weeks of age, they display a modest suppression of weight gain and at 7 weeks of age they show dramatic splenomegaly with the average spleen weight being 10-fold higher than in WT. Splenomegaly persisted in 6- to 8-month-old mice. They have a 17-fold increase in the number of reticulocytes, significant reductions of RBC number, hemoglobin, and hematocrit, along with a dramatic increase in the RBC distribution in FLKO mice. They also have a two- to three-fold increase in the number of circulating white blood cells including lymphocytes, monocytes, neutrophils, and eosinophils. Membrane and cytoskeletal proteins remain at their normal levels, but the major peripheral membrane proteins spectrin, adducin, and actin were greatly reduced. No defects were observed in the heterozygous mice.

Of note, a strain with exons 11 through 15, encoding the carboxy terminal headpiece domain, replaced with a neo cassette is available as Stock No. 034594.

Development

The L1L2_Bact_P cassette was inserted at a position on Chromosome 14 downstream of exon 4 of the dematin actin binding protein (Dmnt) gene. The cassette is composed of a frt site followed by En2SA (mouse engrailed 2 gene splice acceptor), internal ribosome entry site (IRES) fused to a β-galactosidase (lacZ) gene, and a polyadenylation site (SV40 polyA). This was followed by a loxP-flanked cassette containing a neomycin resistance gene under the control of the human beta-actin promoter, SV40 polyA, a second frt site and a second loxP site. A third loxP site is inserted downstream of exon 8. The mutation was generated in C57BL/6N-A^tm1Brd derived JM8A3.N1 embryonic stem (ES) cells, and correctly targeted ES cells were injected into blastocysts. The resulting chimeric mice were bred to C57BL/6J females (Stock No. 000664) and then subsequently mice were backcrossed to C57BL/6J mice for at least 20 generations. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize ROSA26::FLPe knock in oocytes (Stock No. 009086) to make a live colony lacking the frt-flanked lacZ sequence and neomycin cassette. Resulting Epb4.9^fl/fl mice harbor loxP sites flanking exons 5-8.

Allele

Symbol: Dmtn^{tm1c(KOMP)Mbp}
Name: targeted mutation 1c, Mouse Biology Program, UC Davis
MGI accession ID: MGI:6384825
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed)
Synonyms: Epb4.9^fl
Marker symbol: Dmtn
Marker name: dematin actin binding protein
Marker synonyms: dematin; DMT; Epb4.9; EPB49
Chromosome: 14
Strain of origin: C57BL/6N-A^tm1Brd
Molecular note: The L1L2_Bact_P cassette was inserted at a position on Chromosome 14 downstream of exon 4. The cassette is composed of a FRT site followed by En2SA (mouse engrailed 2 gene splice acceptor), internal ribosome entry site (IRES) fused to a beta-galactosidase (lacZ) gene, and a polyadenylation site (SV40 polyA). This was followed by a loxP-flanked cassette containing a neomycin resistance gene under the control of the human beta-actin promoter, SV40 polyA, a second FRT site and a second loxP site. A third loxP site is inserted downstream of exon 8. Flp-mediated recombination removed the FRT-flanked neo cassette leaving exons 5 through 8 floxed.
General note: Personal communication from the investigator, ES cells for Dmtn (Epb4.9) were acquired from KOMP.