CrbnI391V knock-in mice exhibit in vivo sensitivity to thalidomide and its derivatives. They are suitable for use in applications related to the study of B cell malignancies (multiple myeloma, mantle cell lymphoma, chronic lymphocytic leukemia) and thalidomide-induced cytopenias and teratogenicity.
Benjamin Ebert, Dana Farber Cancer Institute
The targeted Crbn gene encodes a substrate recognition component of a DCX (DDB1-CUL4-X-box) E3 protein ligase complex involved in the ubiquitination and subsequent proteasomal degradation of target proteins. Mutations in this gene are associated with autosomal recessive 2 mental retardation and autosomal recessive non-syndromic intellectual disability. These CrbnI391V mice carry an allele for the I391V amino acid substitution mutation which corresponds to the human V388 residue mutation enabling lenalidomide-dependent degradation of casein kinase 1A1 and confers in vivo sensitivity to thalidomide and its derivatives. Sequencing was used to confirm the point mutation. Homozygotes are viable and fertile. T cells from homozygotes exhibit thalidomide derivative (lenalidomide) induced degradation of Ikzf1, Zfp91, and Ck1a (casein kinase 1a1) proteins, and increased secretion of IL-2. Homozygotes treated for 21 days with lenalidomide develop leukopenia, thrombocytopenia, as well as impaired hematopoiesis, specifically reduction in hematopoietic stem and progenitor cell. Heterozygotes display sensitivity to thalidomide and its derivatives. Homozygous mice exposed to thalidomide (or lenalidomide) during pregnancy exhibit fetal loss (at around E10.5), compared to no fetal loss in wildtype controls.
A targeting vector containing a frt and loxP site flanked Neo cassette was utilized in the construction of this mutant. This selection cassette was inserted upstream of exon 11 of the targeted gene along with a point mutation for I391V in exon 11. The construct was electroporated into unspecified C57BL/6 derived embryonic stem (ES) cells. The resulting chimeric animals were tested for germline transmission. The mice were crossed to a FLP recombinase-expressing strain on the C57BL/6 genetic background to excise the Neo selection cassette. The mice were bred to remove the FLP recombinase allele. 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).
In 2021, 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. Four of the 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating, including the rd8 recessive mutation associated with retinal degeneration on Chromosome 1 within the Crb1 locus. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.
|Allele Name||targeted mutation 1.1, Benjamin L Ebert|
|Allele Type||Targeted (Humanized sequence)|
|Gene Symbol and Name||Crbn, cereblon|
|Strain of Origin||C57BL/6|
|Molecular Note||The targeting vector contains an FRT- and loxP-flanked neomycin cassette inserted upstream of exon 11 followed by a point mutation substitution isoleucine for the corresponding human residue, valine (I391V) in exon 11. Flp-mediated recombination removed the FRT-flanked neo cassette.|
When maintaining a live colony, these mice may be bred as homozygotes.
When using the Crbn (I391V), CrbnI391V, CrbnI391V mouse strain in a publication, please cite the originating article(s) and include JAX stock #032487 in your Materials and Methods section.