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.
