P2rx4 tdTomato BAC transgenic mice were designed in the laboratory of Dr. Baljit S. Khakh (University of California Los Angeles).
The ~191 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-448O6 was obtained; containing the entire purinergic receptor P2X, ligand-gated ion channel 4 locus (P2rx4), as well as ~100 kbp 5' (centromeric) flanking region and ~70 kbp 3' (telomeric) flanking region. The centromeric region includes two complete loci - the purinergic receptor P2X, ligand-gated ion channel 7 locus (P2rx7) and microRNA 8115 locus (Mir8115). The telomeric region includes the calcium/calmodulin-dependent protein kinase kinase 2 beta locus (Camkk2).
Using homologous recombination/BAC recombineering, a construct containing a tdTomato cDNA sequence with a BGH polyadenylation sequence was all inserted into the ATG start site of the BAC P2rx4 gene (replacing the translation initiation codon in exon 1). No other loci on the BAC were altered. The tdTomato sequence harbors a rare SbfI restriction site and cutting with SbfI releases a diagnostic 8 kbp band from the P2rx4 tdTomato BAC transgene (also called P2X4 tdTomato, P2X4-tdTomato or P2X4-tdT).
The final ~193 kbp P2rx4 tdTomato BAC transgene was purified and then used for pronuclear injection into eggs derived from a cross of (C57BL/6 x DBA2)F1 hybrids. Founder animals were bred to C57BL/6NTac inbred mice, generating a single founder line with germline transmission. The donating investigator reports that P2rx4 tdTomato BAC transgenic mice were backcrossed ten generations to C57BL/6NTac inbred mice (see SNP notes below), and then hemizygous males with black coat color were sent to The Jackson Laboratory Repository in 2016. Upon arrival, sperm was cryopreserved. To establish our living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6NJ inbred females (Stock No. 005304).
Of note, the donating investigator reports that the Y chromosome may not have been fixed to the C57BL/6N background during backcrossing. The chromosomal insertion site(s) and transgene copy number has not been identified to date (June 2016).
In 2016, a 32 SNP (single nucleotide polymorphism) panel analysis, with 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 males sent to The Jackson Laboratory Repository. All markers throughout the genome suggested a C57BL/6 genetic background with one exception - a chromosome 17 marker (~34 Mbp) was heterozygous for C57BL/6::DBA2. In addition, all 5 markers that determine C57BL/6J from C57BL/6N were found to be C57BL/6N. These data suggest that the mice sent to The Jackson Laboratory Repository are congenic on C57BL/6N, and the transgene insertion site may be on chromosome 17.
