B6.129-Gt(ROSA)26Sor^{tm1(CAG-cas9*,-EGFP)Fezh} Is(14)2Rdf/CjwuJ

Stock number: 037071
Product name: MDRCas9
Strain synonyms: MDRCas9
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

MDRCas9 double mutant mice contain the 14qC3-MDR allele (Stock No. 017642), containing a loxP- and frt-flanked region on Chr 14 that has been implicated in B cell chronic lymphocytic leukemia (CLL). They also contain a Rosa26-LSL-Cas9 knockin allele (Stock No. 026175) with cre recombinase-dependent expression of CRISPR associated protein 9 (cas9) endonuclease and EGFP directed by a CAG promoter. When bred to MDRCD19 double homozygous mice (Stock No. 037070), these mice have applications in studies related to tumorigenesis and therapeutic management of chronic lymphocytic leukemia.

Detailed description

MDRCas9 double mutant mice contain the 14qC3-MDR allele (Stock No. 017642), containing a loxP- and frt-flanked region on Chr 14 that has been implicated in B cell chronic lymphocytic leukemia (CLL) and a downstream GFP fluorescent reporter. They also contain a Rosa26-LSL-Cas9 knockin allele (Stock No. 026175) with cre recombinase-dependent expression of CRISPR associated protein 9 (cas9) endonuclease and EGFP directed by a CAG promoter.

In B cell chronic lymphocytic leukemia the most common genomic aberration is deletion of human chromosomal region 13q14, corresponding to mouse chromosomal region 14qC3. Deletions of this same region are also found in monoclonal B-cell lymphocytosis and diffuse large B-cell lymphomas. An 0.11Mb minimal deleted region (MDR), that includes DLEU2 (deleted in lymphocytic leukemia, 2) and the MIR15A/MIR16-1 cluster, has been identified in more than half the patients with B cell chronic lymphocytic leukemia.

These mice carry the loxP- and frt-flanked MDR allele, 14qC3-MDR. loxP and frt sites flank a 110 kb region that includes the Dleu5, Kcnrg, Dleu2 genes and Mir15a/Mir16-1 cluster. Cre-mediated recombination is indicated by GFP expression.

MDRCas9 mice also carry the Rosa26-LSL-Cas9 knockin allele containing a floxed-STOP cassette preventing expression of the downstream bicistronic sequences of CRISPR associated protein 9 (cas9) endonuclease and EGFP. Although under control of a CAG promoter, widespread expression of cas9 and EGFP is prevented by the STOP cassette. After exposure to Cre recombinase, expression of cas9 and EGFP is observed.

Double homozygous mice are viable and fertile with no discernable disease phenotype. When bred to MDRCD19 double homozygous mice (Stock No. 037070), mice may be useful for studying CLL and Richter syndrome when used in combination with small signal guide RNAs (sgRNAs) by allowing editing of single or multiple mouse genes in vivo or ex vivo. The donating investigator states that this model can be largely applicable to the use of sgRNAs made by their group as well as other novel sgRNAs.

Development

MDRCas9 double mutant mice were generated by crossing 14qC3-MDR mice (Stock No. 017642), containing a floxed region on Chr 14 that has been implicated in B cell chronic lymphocytic leukemia (CLL), to Rosa26-LSL-Cas9 knockin mice (Stock No. 026175). Double homozygous mice were maintained on a C57BL/6 genetic background. Upon arrival at The Jackson Laboratory, 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(CAG-cas9*,-EGFP)Fezh}
Name: targeted mutation 1, Feng Zhang
MGI accession ID: MGI:5583839
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Endonuclease
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: GFP,Green Fluorescent Protein,; cas9,CRISPR associated protein 9,
Site of expression: EGFP and Cas9 will be expressed in cells where promoters driving Cre recombinase are expressed.
Molecular note: The targeting vector inserted into the locus was designed with (from 5' to 3') a 5' homology arm, a ubiquitously expressed CAG promoter, loxP-flanked 3xSV40 polyA stop cassette, a 3X-FLAG epitope tag, a mammalian codon-optimized cas9 gene (derived from Streptococcus pyogenes CRISPR associated protein 9 [SpCas9]) flanked by two nuclear localization signals, a P2A ribosomal skip cleavage peptide sequence, an enhanced Green Fluorescent Protein (EGFP) gene, a woodchuck hepatitis virus post-transcriptional regulatory element, bovine growth hormone polyA, pPGK-Neo-pA positive selection cassette and a 3' homology arm.

Allele

Symbol: Is(14)2Rdf
Name: insertion, Chr 14, Riccardo Dalla-Favera 2
MGI accession ID: MGI:4437013
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Is(14)2Rdf
Marker name: insertion, Chr 14, Riccardo Dalla-Favera 2
Chromosome: 14
Strain of origin: 129S1/Sv-Oca2⁺ Tyr⁺ Kitl⁺
Site of expression: Cre recombination results in EGFP expression in cre-expressing tissues.
Molecular note: LoxP and FRT sites as well as a PGK promoter and Hygro cassette were place about 5 kb telomeric to the alternative exon 1 of Dleu2 by homologous recombination. The targeting was done in ES cells carrying Is(14)1Rdf. The two constructs are in cis configuration.