B6.Cg-Ago2^{tm1.1(DhaA*)Aven}/J

Stock number: 034467
Product name: Ago2^Halo-LSL
Research areas: Cell Biology Research, Developmental Biology Research, Neurobiology Research, Research Tools

Description

Ago2^Halo-LSL knock-in mice have a HaloTag sequence followed by an in-frame loxP-flanked STOP-IRES-FLAG cassette upstream of the Ago2 first coding exon. Prior to introduction of Cre recombinase, this allele expresses a HaloTag protein and a Flag-Ago2 fusion protein. Cre recombinase exposure deletes the loxP-flanked STOP-IRES-FLAG cassette, which results in expression of the Halo-Ago2 fusion protein. These Ago2^Halo-LSL mice allow Halo-enhanced Ago2 pulldown (HEAP) and are useful for identification of miRNA targets (miRNA-mRNA binding sites).

Detailed description

The argonaute protein AGO2 (Eif2c2) binds to miRNAs and target mRNAs, as part of the RNA-induced silencing complex (RISC).

The Ago2^Halo-LSL knock-in allele has a HaloTag sequence that is followed by an in-frame loxP-flanked STOP-IRES-FLAG cassette upstream of the Ago2 first coding exon. The HaloTag encodes a 33 kDa haloalkane dehalogenase that has been mutagenized to form an irreversible covalent bond to synthetic chloroalkane ligands that, when linked to a solid substrate, enable the efficient purification of fusion proteins containing the HaloTag. Prior to introduction of Cre recombinase, the Ago2^Halo-LSL allele expresses a HaloTag protein and a Flag-Ago2 fusion protein. Overall expression may be reduced compared to endogenous Ago2 (see below). The donating investigator reports they do not observe any Halo-Ago2 fusion protein before Cre exposure.

When Ago2^Halo-LSL are bred to mice that express Cre recombinase, the resulting offspring will have the loxP-flanked STOP-IRES-FLAG cassette deleted - placing the HaloTag sequence in-frame with the Ago2 first coding exon and resulting in expression of the Halo-Ago2 fusion protein in the cells/tissues as determined by the Cre promoter. The Halo-Ago2 fusion protein is useful for Halo-enhanced Ago2 pulldown (HEAP) which allows the direct purification of AGO2-containing complexes and the identification of miRNA-mRNA interaction sites - bypassing the need for radiolabeling, immunoprecipitation and gel purification. The Cre-inducible expression of Halo-Ago2 further allows this to be focused to a specific subset of cells - bypassing the need for microdissection and cell purification using cell surface markers.
For example, when Ago2^Halo-LSL are bred to germline Cre-expressing mice (CAGGS-Cre; see Tg(CAG-cre)1Kymm), the resulting Ago2^Halo mice show widespread expression of the Halo-Ago2 fusion protein in all tested tissues (brain, lung, kidney, liver) and mouse embryonic fibroblasts (by PCR and immunoblot analysis). [Li et al. 2019 bioRxiv]

Before introduction of Cre recombinase, mice heterozygous for Ago2^Halo-LSL are viable and fertile with normal breeding, and have no gross physical or behavioral abnormalities. However when breeding heterozygotes together, the resulting homozygous offspring were recovered at sub-Mendelian frequencies (~11.9%, compared to the expected 25%). The donating investigator suggests the reduced viability of homozygous mice might reflect the reduced expression of the Ago2^Halo-LSL allele compared to wildtype Ago2 allele.

Development

The Ago2^Halo-LSL knock-in allele was created in the laboratory of Dr. Andrea Ventura (Memorial Sloan Kettering Cancer Center). First, a targeting vector was designed to insert a HaloTag cDNA sequence (see below), a TEV protease recognition sequence and an in-frame loxP-STOP-IRES-FLAG-loxP cassette upstream of the first coding exon (exon 1), and a frt-flanked PGK-neo cassette in intron 1 of the argonaute RISC catalytic subunit 2 locus (Ago2) on chromosome 15. HaloTag is a 33 kDa haloalkane dehalogenase encoded by the DhaA gene from Rhodococcus rhodochrous that has been mutagenized to form an irreversible covalent bond to synthetic chloroalkane ligands.

The targeting construct was electroporated into (C57BL/6 x 129S4/SvJae)F1-derived v6.5 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeras were bred with C57BL/6J mice for germline transmission. Offspring were bred with β-actin-Flpe mice (Tg(ACTFLPe)9205Dym on a C57BL/6J genetic background; Stock No. 005703) for germline deletion of the PGK-neo cassette. The resulting Ago2^Halo-LSL colony was then backcrossed to C57BL/6J mice for at least seven generations (and the Flpe-expressing allele was removed) prior to sending males to The Jackson Laboratory Repository in 2020. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Of note, the donating investigator reports that, at least once during backcrossing, an Ago2^Halo-LSL female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).

Allele

Symbol: Ago2^{tm1.1(DhaA*)Aven}
Name: targeted mutation 1.1, Andrea Ventura
MGI accession ID: MGI:6390570
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Inserted expressed sequence; Epitope tag
Synonyms: Ago2^Halo-LSL
Marker symbol: Ago2
Marker name: argonaute RISC catalytic subunit 2
Marker synonyms: 1110029L17Rik; 2310051F07Rik; argonaute 2; CASC7; eIF2C 2; Eif2c2; LESKRES; LINC00980; mKIAA1567; PPD; Q10
Chromosome: 15
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Molecular note: The targeting vector is designed to insert a HaloTag cDNA sequence (see below), a TEV protease recognition sequence and an in-frame loxP-STOP-IRES-FLAG-loxP cassette upstream of the first coding exon (exon 1), and an FRT-flanked PGK-neo cassette in intron 1 of the gene. HaloTag is a 33 kDa haloalkane dehalogenase encoded by the DhaA gene from Rhodococcus rhodochrous that has been mutagenized to form an irreversible covalent bond to synthetic chloroalkane ligands. Flp-mediated recombination removed the FRT-flanked neo cassette.