These Mulefl mice possess loxP sites flanking exons 76 and 77 of the Huwe1 gene. This strain may be useful for generating conditional mutations in applications related to the study B cell homeostasis, ubiquitination of apoptotic proteins such as MCL1 and p53 (TRP53).
Dr. Tak Mak, University Health Network/Un of Toronto
The targeted Huwe1 gene encodes an E3 ubiquitin ligase that ubiquitinates MCL1 and p53. Mutations in this gene have been associated with X-linked syndromic mental retardation, Turner type.
These mice possess loxP sites flanking exons 76 and 77 of the targeted gene. Exons 76 and 77 encode the HECT domain, which is critical for E3 ubiquitin ligase activity.
Mice that are homozygous for this allele are viable and fertile. When these mutant mice are bred to mice that express Cre recombinase, resulting offspring will have exons 76 and 77 deleted in the cre-expressing tissues.
Removal of the floxed sequence creates a null allele. During backcrossing, the Y chromosome may not have been fixed to the C57BL/6 genetic background.
When bred to a strain with Cre recombinase expression in B-lymphocytes (see Stock No. 020505 for example), this mutant mouse strain may be useful in studies of B cell homeostasis.
A targeting vector containing a FRT site flanked Neo selection cassette and a 3’ end loxP was utilized in the construction of this mutant. This selection cassette was inserted upstream of exon 76 of the targeted gene, and another loxP site was inserted downstream of exon 77. The construct was electroporated into 129P2/OlaHsd derived E14K embryonic stem (ES) cells.
Correctly targeted ES cells were injected into C57BL/6 blastocysts. The resulting chimeric animals were tested for germline transmission.
Heterozygotes were crossed with Flpe-deleter mice on the B6 genetic background to delete the FRT site flanked NEO cassette. Mice that retained the loxP site flanked exons 76 and 77 were then bred to C57BL/6 mice to remove the FLP, and were backcrossed to C57BL/6 for 6 generations. 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. While the 43 markers throughout the genome suggested a C57BL/6 genetic background, all 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 C57BL/6N genetic background.
|Allele Name||targeted mutation 1.1, Tak Mak|
|Allele Type||Targeted (Conditional ready (e.g. floxed), No functional change)|
|Gene Symbol and Name||Huwe1, HECT, UBA and WWE domain containing 1|
|Strain of Origin||129P2/OlaHsd|
|Molecular Note||A loxP site was inserted upstream of exon 76. An FRT-flanked neo cassette with a 5' loxP site was inserted downstream of exon 77. Flp-mediated recombination removed the neo cassette and left exons 76 and 77 floxed.|
When maintaining a live colony, these mice can be bred as homozygous females and hemizygous males; the gene is X-linked.
When using the Mule flox mouse strain in a publication, please cite the originating article(s) and include JAX stock #029254 in your Materials and Methods section.