B6.Cg-Gt(ROSA)26Sor^{tm1(CAG-GFP/Eif2c2)Zjh}/J

Stock number: 017626
Product name: R26-LSL-tAGO2
Research areas: Neurobiology Research, Research Tools

Description

R26-LSL-tAGO2 mice are used in the miRAP (miRNA tagging and Affinity Purification) method in conjunction with Cre recombinase expressing mice, for genetically targeted microRNA tagging and generating tissue specific miRNA expression profiles.

Detailed description

These mice contain a GFP and myc tagged Eif2c2 gene (tAgo2) under the control of the CAG (cytomegalovirus beta-actin enhancer) promoter, inserted in the Gt(ROSA)26Sor locus. Expression of the GFP/myc tagged Eif2c2 (tAgo2) gene is blocked by a loxP flanked STOP fragment placed between the Eif2c2 sequence and the CAG promoter. The Argonaute protein AGO2 (EIF2C2) binds to miRNAs and target mRNAs, as part of the RNA-induced silencing complex (RISC). The tAgo2 fusion gene was constructed by fusing GFP and myc tags to the N-terminus of Eif2c2. When in the presence of Cre recombinase activity, the GFP/myc tagged Eif2c2 gene is expressed, with GFP expression indicating expression of Eif2c2 and the myc epitope tag allowing affinity purification of associated miRNAs. When crossed to a strain expressing Cre recombinase, miRNAs in cre-expressing cells can be co-immunoprecipitated with tAGO2 by using a MYC antibody. miRNAs from all cells in the tissue sample can be co-immunoprecipitated with AGO2 or tAGO2 by using an AGO2 antibody. Mice that are homozygous for the targeted mutation are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities.

Development

A targeting vector containing loxP site flanked STOP cassette, GFP and myc tagged Eif2c2 coding sequence, under the control of the CAG promoter was inserted in the Gt(ROSA)26Sor locus. The construct was electroporated into C57BL/6 derived Bruce4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts. The resulting chimeric animals were crossed to albino C57BL/6 mice. Heterozygotes were crossed to generate homozygotes. Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-GFP/Eif2c2)Zjh}
Name: targeted mutation 1, Z Josh Huang
MGI accession ID: MGI:5301865
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: B6.Cg-Thy1^a
Expressed genes: GFP,Green Fluorescent Protein,; Ago2,argonaute RISC catalytic subunit 2,mouse, laboratory; myc tag,myc tag,
Site of expression: When bred to mice that express Cre recombinase, EGFP will be expressed in tissues where promoters driving Cre recombinase are expressed.
Molecular note: A targeting vector containing loxP site flanked STOP cassette, GFP, and myc-tagged Eif2c2 coding sequence under the control of the CAG promoter was inserted in the Gt(ROSA)26Sor locus. When in the presence of Cre recombinase activity, the GFP/myc tagged Eif2c2 gene is expressed, GFP expression indicates expression of Eif2c2, and the myc epitope tag allowing affinity purification of associated miRNAs.