B6.Cg-Gt(ROSA)26Sor^{tm1(SPOP*F133V)Mrbn}/J

Stock number: 030102
Product name: R26^F133V
Research areas: Developmental Biology Research, Internal/Organ Research, Reproductive Biology Research, Research Tools

Description

Expression of mutant SPOP-F133V protein is prevented in these R26^F133V mice until crossed to a strain expressing Cre recombinase under the control of a promoter of interest. This strain may be useful when studying prostate cancer.

Detailed description

R26^F133V mice contain a loxP-flanked STOP sequence upstream of a mutated human speckle-type POZ protein (SPOP) gene. SPOP encodes for the substrate recognition/binding protein of the E3 ubiquitin ligase complex. Mutations in SPOP are the most common point mutations in primary prostate cancer with characteristic genomic alterations, patterns of genomic rearrangements, gene expression profiles and methylation patterns. SPOP mutations occur in approximately 10% of patients with localized disease. The SPOP-F133V mutation present in these mice results in a missense mutation. Widespread expression of SPOP-F133V is prevented by a strong transcriptional stop sequence. When bred to mice that express Cre recombinase under the control of a promoter of interest, the loxP-flanked STOP sequence is removed, and SPOP-F133V-IRES-EGFP is expressed in cre-expressing cells. Homozygous mice are viable and fertile.

When bred to STOCK Tg(Pbsn-cre)4Prb/J mice (Stock No. 026662), resulting mice express mutant SPOP-F133V in epithelial cells of the prostate. By one year of age focal area with histological atypia is detectable. EGFP expression is only detected via immunohistochemistry.

Development

A targeting vector was designed placing a loxP-flanked STOP cassette upstream of the human speckle-type POZ protein (SPOP) gene. A mutation was inserted at amino acid 133, resulting the missense mutation, F133V, in the substrate binding MATH domain. This mutation is homologous to the F133V mutation found in patients with prostate cancer. The human SPOP-F133V sequence has a Flag tag followed by a Myc sequence. An internal ribosome entry site (IRES) fused to an enhanced green fluorescent protein (EGFP) and a polyadenylation sequence were inserted downstream of the SPOP sequence. This construct was inserted into the Gt(ROSA)26Sor locus, between exon 1 and 2, via electroporation into B6(Cg)-Tyr^c-2J/J-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6J blastocysts. Chimeric males were bred to albino B6 females. The resulting R26^F133V males were bred to C57BL/6J females, and were maintained on a C57BL/6J 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).

Allele

Symbol: Gt(ROSA)26Sor^{tm1(SPOP*F133V)Mrbn}
Name: targeted mutation 1, Mark A Rubin
MGI accession ID: MGI:5882069
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Inserted expressed sequence; Humanized sequence
Synonyms: R26^F133V
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: C57BL/6
Expressed genes: SPOP,speckle type BTB/POZ protein,human
Site of expression: When bred to mice that express Cre recombinase under the control of a promoter of interest, the loxP-flanked STOP sequence is removed, and SPOP-F133V-IRES-EGFP is expressed in cre-expressing cells.
Molecular note: The targeting vector introduced a conditional ready flag-myc tagged human gene with the valine for phenylalanine at position 133 (F133V) mutation followed by an IRES and EGFP reporter gene with a nuclear localization sequence.