B6N.Cg-Tg(Camk2a-P2rx2/YC3.1)21Khakh/J

Stock number: 025114
Product name: SPRAE
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

SPRAE mice have mossy fiber expression of functional P2X2 receptors tagged with a FRET-based Ca2+ sensor (P2X2-YC), allowing fluorescent imaging of the location, regional expression, mobility and activation of transmitter-gated P2X channels. These mice are used to image P2X2 receptor activation and to selectively drive glutamate release in mossy fiber terminals for studing astrocyte signaling.

Detailed description

P2X2 receptors are cell-surface ATP-gated Ca2+ permeable cation channels in presynaptic terminals and axons that, when activated, trigger neurotransmitter release from mossy fibers; "spraying" the stratum lucidum region with glutamate.

These "Selective P2X Receptor Axoterminal Excitation” (SPRAE) mice harbor the CaMKIIα-P2X2-YC BAC transgene designed for mossy fiber expression of P2X2 receptors with fluorescent YC3.1 tags on tolerant positions of their cytosolic domain. P2X2-YC receptors are functional and show ATP-evoked currents and FRET signals (strong yellow and weaker cyan fluorescence). The donating investigator reports YFP and CFP expression are evident by direct fluorescent measurement, but immunohistochemistry may be hampered if antibodies are unable to discriminate YFP from CFP. Hemizygous and homozygous mice are reported to be viable and fertile.

The P2X2-YC fusion gene (also called P2X₂-cam) encodes the rat P2X2 receptor (P2rx2) fused at its carboxy terminus (cytosolic domain) to the fluorescence resonance energy transfer (FRET)-based optical reporter Yellow Cameleon 3.1 (YC3.1). YC3.1 contains a Ca2+ sensor domain fused between a FRET chromophore pair (yellow fluorescent protein [YFP] donor / cyan fluorescent protein [CFP] acceptor). Upon Ca2+ binding (channel opening) a conformational change in the sensor domain alters YFP-CFP proximity and results in increased FRET; this can be used to measure relatively large Ca2+ transients that are above background. P2X2-YC has a Ca2+ affinity in the micromolar range (~2 μM) and FRET efficiency of ~24.5%. Each P2X2 receptor carries three YC3.1 tags.

Development

The CaMKIIα-P2X2-YC BAC transgene was designed in the laboratory of Dr. Baljit S. Khakh (University of California Los Angeles). First, the ~221 kbp mouse bacterial artificial chromosome (BAC) RP23-221H5 was obtained, containing the entire Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) locus, ~110 kbp 5prime flanking region (including the Arsi locus and a portion of the Tcof1 locus) and ~50 kbp 3prime flanking region (including the Slc6a7 and Cdx1 loci). The P2X2-YC fusion gene (described below) was inserted immediately upstream of the CaMKIIα translation initiation codon in exon 1 on the RP23-221H5 BAC via homologous recombination/BAC recombineering.

The CaMKIIα-P2X2-YC BAC was purified and then used for pronuclear injection into B6D2F1 embryos. The resulting CaMKIIα-P2X2-YC BAC transgenic founder mice were bred with C57BL/6NTac mice. Transgenic mice from founder line Tg21 were found with expression of fluorescently-tagged P2X2 receptors within mossy fibers. The donating investigator reported that these "SPRAE" mice (Selective P2X Receptor Axoterminal Excitation) were bred with C57BL/6NTac wildtype mice for several generations (see SNP results below) prior to sending to The Jackson Laboratory Repository in 2014. Upon arrival, sperm was used to fertilize C57BL/6NJ oocytes (Stock No. 005304). The first generation rederived mice were then bred to C57BL/6NJ at least one generation. After at least one more generation of breeding the colony with C57BL/6NJ mice, sperm will be cryopreserved from SPRAE animals.

A 32 SNP panel analysis (27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains) was performed on the first generation rederived living colony at The Jackson Laboratory Repository. This revealed 3 of 27 markers, one each on chromosomes 1, 12 and 15, that were not fixed for C57BL/6 allele-type (e.g., still segregating for DBA/2 allele-type markers). All 5 markers that determine C57BL/6J from C57BL/6N were found to be C57BL/6N allele-type. Taken together, these data suggest the mice sent to The Jackson Laboratory Repository were not completely backcrossed onto C57BL/6N (i.e., were a mix of C57BL/6N [~78%] and DBA/2 [~22%]). Because The Jackson Laboratory Repository bred the first generation rederived mice to C57BL/6NJ at least one generation, the live colony as of April 2015 is estimated to be a mix of C57BL/6N (~94%) and DBA/2 (~6%). Further generations of breeding the colony with C57BL/6NJ mice will result in the incipient congenic SPRAE strain.

The P2X2-YC fusion gene (also called P2X₂-cam) encodes the rat P2X2 receptor (P2rx2) fused at its carboxy terminus (cytosolic domain) to the fluorescence resonance energy transfer (FRET)-based optical reporter Yellow Cameleon 3.1 (YC3.1). YC3.1 contains a Ca2+ sensor domain fused between a FRET chromophore pair (yellow fluorescent protein [YFP] donor / cyan fluorescent protein [CFP] acceptor). Upon Ca2+ binding (channel opening) a conformational change in the sensor domain alters YFP-CFP proximity and results in increased FRET; this can be used to measure relatively large Ca2+ transients that are above background. P2X2-YC has a Ca2+ affinity in the micromolar range (~2 μM) and FRET efficiency of ~24.5%. Each P2X2 receptor carries three YC3.1 tags.

Allele

Symbol: Tg(Camk2a-P2rx2/YC3.1)21Khakh
Name: transgene insertion 21, Baljit Khakh
MGI accession ID: MGI:5569123
Generation method: Transgenic
Attributes: Reporter; Inserted expressed sequence
Marker symbol: Tg(Camk2a-P2rx2/YC3.1)21Khakh
Marker name: transgene insertion 21, Baljit Khakh
Chromosome: UN
Strain of origin: (C57BL/6 x DBA/2)F1
Expressed genes: P2rx2,purinergic receptor P2X 2,rat; YFP,Yellow Fluorescent Protein,jellyfish; CFP,Cyan Fluorescent Protein,jellyfish
Site of expression: Astrocytes in the hippocampal mossy fiber pathway.
Molecular note: The transgene contains the entire Ca2+/calmodulin-dependent protein kinase II alpha locus, ~110 kbp 5' flanking region (including the Arsi locus and a portion of the Tcof1 locus) and ~50 kbp 3' flanking region (including the Slc6a7 and Cdx1 loci) from BAC RP23-221H5. The P2X2-YC3.1 fusion gene was inserted immediately upstream of the CaMKIIalpha translation initiation codon in exon 1 via homologous recombination/BAC recombineering. The P2rx2/YC3.1 fusion gene encodes rat P2rx2 fused at its carboxy terminus to the fluorescence resonance energy transfer (FRET)-based optical reporter Yellow Cameleon 3.1 (YC3.1).