B6.Cg-Gria2^{em1(DhaA*)Sprus}/J

Stock number: 041117
Product name: GluA2-HaloTag, GluA2-HT

Research areas: Research Tools

Description

GluA2-HaloTag (GluA2-HT) knock-in mice express a HaloTag (HT) protein from the mouse Gria2 promoter, enabling the mapping of learning-related synaptic plasticity on a brain-wide scale.

Detailed description

Turnover of the ioniotropic glutamate receptor subunit GluA2, encoded by Gria2 (glutamate receptor, ionotropic, AMPA2 (alpha 2)), is an indicator of synaptic plasticity.

GluA2-HaloTag (GluA2-HT) knock-in mice, expressing a HaloTag (HT) protein fused to the extracellular side of GRIA2, enable the mapping of learning-related synaptic plasticity on a brain-wide scale. Protein expression levels of Gria2 are not affected by the presence of the mutation. HaloTag encodes 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. When linked to a solid substrate, it enables the efficient purification of fusion proteins containing the HaloTag.

DELTA (Dye Estimation of the Lifetime of proTeins in the brAin) is a new method for mapping brain-wide changes in synaptic protein turnover with single-synapse resolution, based on Janelia Fluor (JF) dyes and HaloTag (HT) knock-in mice. DELTA measures protein dynamics in vivo using pulse-chase experiments with spectrally separable fluorescent ligands. It enables precise measurements of protein lifetime in the whole brain and other tissues in vivo, down to synaptic resolution.

In DELTA testing of a visually guided foraging task, GluA2 turnover in homozygous GluA2-HaloTag mice was found to be elevated in hippocampal CA1, with higher GluA2 turnover in dendrites receiving input from hippocampal CA3 compared to entorhinal cortex. A more widespread pattern of synaptic protein turnover was seen in response to environmental enrichment.

Development

The GluA2-HT knock-in line was generated by CRISPR/Cas9 pronuclear microinjection. The HaloTag (HT) gene was inserted after the signal peptide encoded by exon 1 of the Gria2 (GluA2) gene on mouse Chromosome 3 and flanked by two linkers, GGGGSGGGS at the 5' end and GGGGSGGGSGGGGSGGGS at the 3' end (GRCm38). The knock-in DNA was co-injected with a gRNA (5'-TCTTCTAACAGCATACAGATAGG-3') and Cas9 protein (Fisher Scientific, A36498) into B6D2F1/J mouse one-cell embryos. Resultant mice were backcrossed to C57BL/6J for 5 generations by the donating laboratory. This is founder line 1. Upon arrival, sperm was cryopreserved. To establish a live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Gria2^{em1(DhaA*)Sprus}
Name: endonuclease-mediated mutation 1, Nelson Spruston
MGI accession ID: MGI:8246868
Generation method: Endonuclease-mediated
Attributes: Epitope tag
Synonyms: GluA2-HT
Marker symbol: Gria2
Marker name: glutamate receptor, ionotropic, AMPA2 (alpha 2)
Marker synonyms: GluA2; GLUR2; Glur-2; GLURB; GluR-B; GluR-K2; HBGR2; NEDLIB
Chromosome: 3
Strain of origin: (C57BL/6J x DBA/2J)F1
Molecular note: Using CRISPR/Cas9 technology, HaloTag (HT) gene was inserted after the signal peptide encoded by exon 1 of the Gria2 (GluA2) gene and flanked by two linkers, GGGGSGGGS at the 5' end and GGGGSGGGSGGGGSGGGS at the 3' end. 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.