B6;129P2-Fadd^tm1Mak/J

Stock number: 029255
Product name: Fadd Knock-out
Research areas: Apoptosis Research, Developmental Biology Research, Research Tools

Description

This Fadd knockout strain may be useful in studies of apoptosis and immune response involving interferon signaling.

Detailed description

The targeted Fadd gene encodes an apoptosis adaptor protein with a C-terminal death domain that is involved in signaling between death receptors and caspases, as well as early T cell development. Mutations in this gene have been associated with fadd-related immunodeficiency. These knock-out mice carry an allele in which the entire coding region of the Fadd gene has been replaced by a PGK-Neo cassette in opposite orientation. No gene product (protein) is detected by Western blot analysis of total protein lysates from homozygotes. Mice that are heterozygous for this allele are viable and fertile. Homozygotes exhibit an embryonic lethal phenotype, failing to survive past embryonic day E12.5. Most homozygous embryos are smaller in size than controls, and exhibit thin ventricular myocardium and under-developed inner trabeculation by E10.5. By E11.5, approximately 50% homozygous embryos are hemorrhagic. Primary embryonic fibroblasts isolated from homozygotes show impaired TNF-induced endosomal acid sphingomyelinase activation, and are not induced into apoptosis by FAS (CD95), TNFRSF1A (TNF-R55), and TNFRSF25 (DR3). During backcrossing, the Y chromosome may not have been fixed to the C57BL/6 genetic background.

Development

A targeting vector containing a PGK-Neo (in reverse orientation) was used to disrupt the entire coding region of the Fadd gene (2 exons). The construct was electroporated into 129P2/OlaHsd derived E14K embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6J blastocysts. The resulting chimeric males animals were bred to C57BL/6J females. The mice were backcrossed to C57BL/6 for 8 generations by the donating lab (see SNP note below). During backcrossing, the Y chromosome may not have been fixed to the C57BL/6 genetic background. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

A 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. Three of the 43 markers throughout the genome were segregating, suggesting an incomplete backcross. Also, 4 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.

Allele

Symbol: Fadd^tm1Mak
Name: targeted mutation 1, Tak W Mak
MGI accession ID: MGI:2159360
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Fadd
Marker name: Fas (TNFRSF6)-associated via death domain
Chromosome: 7
Strain of origin: 129P2/OlaHsd
Molecular note: A neomycin selection cassette replaced a genomic fragment containing the entire coding region of the gene. Western blot analysis on lysates derived from homozygous mice confirmed that no detectable protein was expressed from this allele.
General note: Authors in J:46527 state that injection of mutant ES cells into Gt(ROSA)26Sor E3.5 blastocysts (J:79478) resulting in high percentage chimeric embryos with a high contribution of mutant cells to the heart reproduce the phenotype of homozygous mutant embryos derived from heterozygous breedings. In contrast, chimeric embryos with a low contribution from the mutant ES cells display a normal phenotype at E11.5.