C57BL/6-Gt(ROSA)26Sor^{tm1(CAG-GFP,-Mars*L274G)Esm}/J

Stock number: 028071
Product name: STOPflox R26-MetRS*
Strain synonyms: FUNCAT
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

This STOPflox R26-MetRS* knock-in strain enables genetically targeted (Cre-dependent) metabolic labeling of endogenous proteins in specific cell-types in vivo for subsequent imaging or purification and biochemical analysis.

Detailed description

The L274G point mutation in methionyl-tRNA synthetase results in an altered binding site that enables loading of non-canonical amino-acid methionine analogs, such as azidonorleucine (ANL), onto methionine tRNA. As endogenous wildtype methionyl-tRNA synthetase does not bind ANL, expression of the mutant MetRS (MetRS*) allows the incorporation of ANL into nascent proteins which can then be subsequently labeled by fluorescent non-canonical amino-acid tagging (FUNCAT) or bioorthogonal non-canonical amino acid tagging (BONCAT) for imaging or purification, respectively. STOPflox R26-MetRS* mice were generated by knocking in a floxed STOP cassette, GFP, 2A sequence and mutant Mars sequence, all under the control of a CAG promoter, into the endogenous Gt(ROSA)26Sor locus. The 2A sequence enables expression of the GFP and mutant Mars as discrete translation products from the same single promoter. Following Cre recombinase mediated removal of the floxed STOP cassette, GFP expression and ANL incorportation are only detected in cells expressing the mutant methionine-tRNA synthetase, MetRS* (Mars*). Mice that are homozygous for the knock-in allele are viable and fertile.

When bred to a strain with Cre recombinase expression in CA1 pyramidal cell layer in the hippocampus of the forebrain (see Stock No. 005359 for example), this mutant mouse strain may be useful in studies of nascent proteins and protein synthesis in CA1 pyramidal neurons.

Development

An exchange vector was designed to contain a mutant F3 FRT site, a NEO selection cassette, CAG promoter, a floxed STOP cassette, Green Fluorescent Protein coding sequence, foot-and-mouth disease virus 2A peptide sequence, the mouse methionine-tRNA synthetase (Mars) with the L274G point mutation, and a FRT site, for recombinase mediated cassette exchange (RMCE). This vector was integrated into the Gt(ROSA)26Sor locus by RMCE. The construct was electroporated into C57BL/6NTac derived A-A02 embryonic stem (ES) cells (Taconic GmbH). Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were crossed to C57BL/6 mice. Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to before 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-GFP,-Mars*L274G)Esm}
Name: targeted mutation 1, Erin M Schuman
MGI accession ID: MGI:6095164
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inserted expressed sequence
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: C57BL/6NTac
Expressed genes: GFP,Green Fluorescent Protein,; Mars1,methionine-tRNA synthetase 1,mouse, laboratory
Site of expression: Following Cre recombinase mediated removal of the floxed STOP cassette, GFP expression and ANL incorportation are only detected in cells expressing the mutant methionine-tRNA synthetase, MetRS* (Mars*).
Molecular note: The exchange vector is designed to contain a mutant F3 FRT site, a NEO selection cassette, CAG promoter, a floxed STOP cassette, Green Fluorescent Protein coding sequence, foot-and-mouth disease virus 2A peptide sequence (to mediate ribosomal skipping), the mouse methionine-tRNA synthetase (Mars) with the L274G point mutation, and an FRT site. This vector was integrated into the Gt(ROSA)26Sor locus by recombinase mediated cassette exchange (RMCE). The L274G mutation allows the binding of the methionine surrogate azidonorleucine (ANL) onto the methionine tRNA. ANL can be tagged for imaging or affinity purification.
General note: Embryonic stem cells = A-A02