B6.Cg-Mapkapk2^tm1.1Yaff/J

Stock number: 024176
Product name: MK2^CV
Research areas: Apoptosis Research, Cancer Research, Cell Biology Research, Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

MK2^CV has exon 2 of the MAP kinase-activated protein kinase 2 gene flanked with loxP sites in opposing orientations. A single exposure to Cre recombinase inverts exon 2 - this generates a STOP codon and results in a functional null allele. In addition, exon 2 can switch reversibly between the MK2-expressing and MK2-null orientation as long as Cre recombinase is active in the cell. These MK2^CV mice may be useful in studying the p38 MAP kinase/MK2 pathway, other checkpoint control pathways, DNA damage response, DNA repair, cell cycle arrest and apoptosis - as well as with other cancer models to study sensitization of tumors to DNA-damaging chemotherapeutics in vivo.

Detailed description

The MK2^CV allele has exon 2 of the MAP kinase-activated protein kinase 2 gene flanked with inward-facing loxP sites (loxP sites in opposing orientations). Homozygous mice are viable and fertile. Prior to Cre recombinase exposure, exon 2 is in the wildtype configuration (MK2^+CV); resulting in normal MK2-expression. A single exposure to Cre recombinase creates the inverted exon 2 configuration (MK2^-CV); the resulting transcript has a STOP codon in the beginning of exon 3 that encodes a highly truncated reading frame that is not translated into functional MK2 proteins (i.e., loss of both the 46 KDa and 42 KDa isoforms corresponding to Uniprot P49137-1 and P49137-2 for human MK2). Because inward-facing loxP sequences result in cre-mediated inversion rather than excision, exon 2 can reversibly switch from the MK2-expressing orientation (MK2^+CV) to the MK2-null orientation (MK2^-CV) and back as long as Cre recombinase is active in the cell. In addition, the MK2 expression state that is established by a single Cre recombinase pulse (e.g., germline cre expression or transient cre expression via adenoviral injection) within the cell is maintained throughout further daughter cell generations.

These MK2^CV mice may be useful in studying the p38 MAP kinase/MK2 pathway, other checkpoint control pathways, DNA damage response, DNA repair, cell cycle arrest and apoptosis; as well as with other cancer models to study sensitization of tumors to DNA-damaging chemotherapeutics in vivo.

For example, when MK2^CV mice are bred to harbor the Kras^LSL-G12D and p53^flox alleles (see Stock Nos. 008179 and 008462, respectively), administration of a Cre recombinase-expressing adenovirus generates mosaic MK2-deficient or MK2-proficient non-small cell lung adenocarcinomas in the same animal. This allows one to study the role of MK2 in cancer development, progression, and response to DNA-damaging chemotherapy in non-small cell lung cancer.

Of note, The Jackson Laboratory Repository also has available a "traditional" floxed allele (loxP sites in same orientation) designed to eliminate Mapkapk2 exon 2 permanently upon exposure to Cre recombinase (MK2^FL; Stock No. 032450).

Development

A targeting vector created by Dr. Michael B. Yaffe (Massachusetts Institute of Technology) was designed to insert "inward-facing" loxP sites flanking exon 2 of the MAP kinase-activated protein kinase 2 gene (Mapkapk2). Sequence analysis confirmed the 5' loxP site is inverted (i.e., in reverse complement sequence to the 3' loxP site) [February 2019]. The targeting vector also placed a frt:PGK-neo:frt sequence downstream of the second loxP site. This construct was electroporated into (C57BL/6 x 129S4/SvJae)F1-derived v6.5 embryonic stem (ES) cells. Chimeric males were bred with ACTB-FLPe transgenic mice (backcrossed eight generations onto C57BL/6J) to eliminate the frt-flanked PGK-neo cassette. The resulting mice with inward-facing loxP sites flanking exon 2 (and a single frt site remaining downstream of exon 2) were identified and termed MK2 "Cre-versible" mice (or MK2^CV mice). The MK2^CV colony was then backcrossed to C57BL/6J inbred mice for ten generations (and the FLPe transgene was removed) prior to sending to The Jackson Laboratory Repository in 2014. Upon arrival, sperm was cryopreserved. To generate the living MK2^CV colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J female mice (Stock No. 000664). The MK2^CV colony was then maintained by breeding heterozygous mice with wildtype mice from the colony or with C57BL/6J inbred mice.

Allele

Symbol: Mapkapk2^tm1.1Yaff
Name: targeted mutation 1.1, Michael Yaffe
MGI accession ID: MGI:5528531
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Mapkapk2
Marker name: MAP kinase-activated protein kinase 2
Chromosome: 1
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Molecular note: A targeting vector was designed to insert a loxP site upstream of exon 2, and a second loxP site (in reverse complement sequence to the first loxP site) downstream of exon 2 as well as a frt:PGK-neo:frt sequence downstream of the second loxP site. Because inward-facing loxP sequences result in cre-mediated inversion rather than excision, exon 2 can reversibly switch from Mapkap2 expression orientation (MK2<+CV>, CV; Cre-versible) to the null orientation (MK2<-CV>) and back as long as Cre recombinase is active in the cell.FLP recombinase mediated recombination removed the neomycin cassette.