C57BL/6-Gt(ROSA)26Sor^{tm2(CAG-EGFP,-Mars1*L274G)Esm}/J

Stock number: 040661
Product name: 3xMetRS*
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

This STOPflox R26-3xMetRS* 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

3xMetRS* is an improved version of STOPflox R26-MetRS* (1xMetRS*; Stock No. 028071) carrying three copies of methionyl-tRNA synthetase 1 with L274G point mutation (Mars1). The mutation results in an expanded binding site that enables loading of the non-canonical amino-acid methionine analogs, azidonorleucine (ANL), onto methionine tRNA. As endogenous wildtype methionyl-tRNA synthetase does not bind ANL, only expression of the mutant MetRS (MetRS*) allows the incorporation of ANL into nascent proteins. They 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 R26-3xMetRS* mice were generated by knocking in a floxed STOP cassette, three copies of the mutant Mars1 sequence, and one copy of EGFP per allele, all under the control of a CAG promoter with all four parts separated by different self-cleaving 2A peptides, into the endogenous Gt(ROSA)26Sor locus. The 2A sequence enables the expression of the three copies of mutant Mars1 and EGFP as discrete translation products from a single promoter. Following Cre recombinase-mediated removal of the floxed STOP cassette, EGFP expression and ANL incorporation are only detected in cells expressing the mutant methionine-tRNA synthetase, MetRS* (Mars1*). Mice that are heterozygous or homozygous for the knock-in allele are viable and fertile. Both heterozygous and homozygous mice showed no obvious behavioral differences with wild-type mice.

Compared to 1xMetRS* (Stock No. 028071), the 3xMetRS* mouse line allows faster and more efficient labelling with the non-canonical amino acid ANL and does not need a low methionine diet for labelling. It works for labelling in the heterozygous state (3xMetRS*) as well as the homozygous state (6xMetRS*) when activated by Cre-mediated excision of the floxed STOP. When bred to a strain with cell type-specific Cre recombinase expression, it can be used for studies on cell type-specific proteomes, protein synthesis, or degradation in any tissue.

Of note, strains containing mutant aminoacyl-tRNA synthetases that recognize azide-bearing noncanonical amino acids include:
MetRS* (Stock No. 028071), PheRS* (Stock No. 033734), and TyrRS* (Stock No. 033735).

MetRS* (Stock No. 028071) is the original strain with single 1xMetRS*.

Development

An exchange vector was used for recombinase mediated cassette exchange (RMCE) that contained (from 5' to 3') a mutant F3 FRT site, a NEO selection cassette, CAG promoter, loxP site, 1200 bp STOP cassette (transcriptional stop codons in all three reading frames and hGH polyA signal), loxP site, three copies of the mouse methionine-tRNA synthetase 1 (Mars1) cDNA with L274G point mutation (each copy followed by T2A, P2A, E2A respectively), an Enhanced Green Fluorescent Protein (EGFP) coding sequence, WPRE, human growth hormone polyA, and a FRT site. This vector was integrated into the Gt(ROSA)26Sor locus by RMCE. The construct was electroporated into C57BL/6NTac-derived embryonic stem (ES) cells harboring RMCE docking sites at the Gt(ROSA)26Sor locus. A correctly targeted ES cell clone was injected into blastocysts. Sperm from the resulting chimeric animals were used for IVF with C57BL/6NTac oocytes. The heterozygous offspring were bred to homozygosity. The donating laboratory maintained the colony via homozygous breeding, with intermittent crosses to C57BL/6JCrl mice [but less than 5 generations total to prevent genetic drift] prior to sending homozygous males with black coat color to The Jackson Laboratory in 2025. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

Allele

Symbol: Gt(ROSA)26Sor^{tm2(CAG-EGFP,-Mars1*L274G)Esm}
Name: targeted mutation 1, Erin M Schuman
MGI accession ID: MGI:8252910
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inserted expressed sequence
Synonyms: 3xMetRS*
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/6NTac
Molecular note: The allele consists of the CAG, floxed STOP-MetRS(L274G)-T2A-MetRS(L274G)-P2A-MetRS(L274G)-E2A-EGFP (WPRE ) inserted into the mouse ROSA26 locus using recombination-mediated cassette exchange in embryonic stem cells. The vector is designed to contain a mutant F3 FRT site, a NEO selection cassette, CAG promoter, a floxed STOP cassette, triple repeat of the mouse methionine-tRNA synthetase (Mars1) with the L274G point mutation separated by T2A, P2A, followed by E2A peptide sequence (to mediate ribosomal skipping) followed by Green Fluorescent Protein coding sequence, WPRE 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? made for us by Taconic GmbH