B6(129S4)-Nras^tm1.1Kshn/J

Stock number: 035733
Product name: Nras^{LSL-G12D/C181S}
Research areas: Cancer Research, Research Tools

Description

Nras^G12D,C181S mice carry the point mutations G12D in exon 1 and C181S in exon 5 of the neuroblastoma ras oncogene (Nras) gene, expression of which is blocked by the presence of a loxP-flanked STOP cassette. When bred to a strain expressing Cre recombinase under control of various tissue specific promoters, Cre recombination deletes the LSL cassette and allows the expression of the mutant NRAS oncogenic protein. This strain may be useful in studying cancer and development, such as palmitoylation cycle in NRAS mutant blood cancers and myeloid transformation.

Detailed description

The Nras^G12D,C181S allele has a loxP-flanked STOP cassette inserted upstream of exon 1 of the endogenous neuroblastoma ras oncogene (Nras) gene. Downstream of this cassette, a guanine to adenine mutation at codon 12 in exon 2 was introduced, resulting in a glycine to aspartic acid mutation (G12D). Also, a guanine to cytosine mutation at codon 181 in exon 5 was introduced, resulting in a cysteine to serine mutation (C181S).

Ras genes are small GTPases that act downstream of receptors and nonreceptor tyrosine protein kinases in multiple pathways to transfer information to the nucleus. Nras mutations have been found to be associated with human somatic rectal cancer, follicular thyroid cancer, autoimmune lymphoproliferative syndrome, Noonan syndrome, and juvenile myelomonocytic leukemia.

Double homozygous Nras^G12D,C181S mice are viable and fertile. The STOP cassette prevents the expression of mutant Nras until it is removed by Cre-mediated recombination of the loxP sites, thus allowing expression of Nras^G12D,C181S.

When bred to Mx1-Cre mice (Stock No. 003556), and induced with poly I:C to drive expression of Cre recombinase at weaning, homozygous and heterozygous offspring do not develop the hematologic neoplasms seen in Nras^G12D. Heterozygous Nras^{G12D,C181S/WT} mice exhibit normal patterns of hematopoiesis, with normal spleen sizes and normal distribution of hematopoietic stem and progenitor cells (HSPCs) and mature blood cells. Homozygous Nras^G12D,C181S mice exhibit suppressed myeloid cell transformation.

Development

The Nras^C181S targeting vector was designed to insert a frt-flanked neomycin resistance (neo) cassette downstream of exon 5 of the neuroblastoma ras oncogene (Nras) gene. Endogenous exon 5 was modified to harbor a guanine to cytosine mutation at codon 181, resulting in a cysteine to serine mutation (C181S). The construct was electroporated into C57BL/6-derived V26.2 embryonic stem (ES) cells previously targeted to contain the LSL-Nras^G12D mutation present in Stock No. 008304. The LSL-Nras^G12D allele consists of a floxed STOP cassette upstream of exon 1 and a guanine to adenine mutation at codon 12 in exon 2, resulting in a glycine to aspartic acid mutation (G12D). Correctly targeted ES cells were injected into albino C57BL/6 blastocysts and resulting chimeric mice were mated to flpo deleter mice (Stock No. 012930) to remove the frt-flanked neo cassette. Resulting Nras^G12D,C181S mice were crossed to remove the flp-expressing transgene. Mice were subsequently bred to C57BL/6J mice (Stock No. 000664) for at least 9 generations. Upon arrival at The Jackson Laboratory Repository, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

Allele

Symbol: Nras^tm1.1Kshn
Name: targeted mutation 1.1, Kevin Shannon
MGI accession ID: MGI:6469907
Generation method: Targeted
Attributes: Not Specified
Marker symbol: Nras
Marker name: neuroblastoma ras oncogene
Chromosome: 3
Strain of origin: C57BL/6
Molecular note: The targeting vector is designed to insert an FRT-flanked neomycin resistance (neo) cassette downstream of exon 5. Endogenous exon 5 was modified to harbor a guanine to cytosine mutation at codon 181, resulting in a cysteine to serine mutation (C181S). This targeting vector then was used to retarget Nras^tm1Tyj inserting C181S in cis to the existing G12Dmutation. Flp-mediated recombination removed the FRT-flanked neo cassette.