B6N.Cg-Tg(P2rx4-tdTomato)1Khakh/J

Stock number: 029332
Product name: P2rx4 tdTomato BAC transgenic
Research areas: Neurobiology Research, Research Tools

Description

P2rx4 tdTomato BAC transgenic mice exhibit widespread distribution of cell populations throughout the brain expressing tdTomato fluorescence, consistent with that expected for P2X4 expression. These mice are a fluorescent tool useful in studying guided electrophysiology on P2RX4-expressing cells, as well as for ATP signaling throughout the body in organs such as brain, blood, heart, gut and lung.

Detailed description

P2rx4 tdTomato BAC transgenic mice express the orange/red fluorescent protein tdTomato under control of the P2rx4 locus promoter/enhancer regions within the BAC transgene. P2rx4 tdTomato transgenic mice exhibit widespread distribution of tdTomato-expressing cells throughout the brain in a pattern that faithfully represents P2X4 expression. The tdTomato expression is visible via native/direct fluorescence, although the use of antibody staining/immunohistochemistry would assist visualization of low-expressing cell types.

Specifically, the highest tdTomato expression is abundant and strong in glomerular layer of olfactory bulb and arcuate nucleus of hypothalamus. High expression is seen in molecular layer of cerebellum, suprachiasmatic and paraventricular nuclei of hypothalamus, locus coeruleus of hindbrain and several ventricular structures. Moderate-to-low expression is observed in other regions of the olfactory bulb, cerebral cortex, subcortical telencephalon (CA1-CA4), cerebellum (purkinje cells), hypothalamus, thalamus, midbrain and hindbrain. In the retina, we see tdTomato expression in amacrine cells and horizontal cells. Little-to-no expression is shown in dentate gyrus (subcortical telencephalon). The tdTomato expression in peripheral organs (such as lung and heart) has not been characterized to date (June 2016).

Regarding presence of the P2rx7 locus on the BAC transgene, the primary publication states they found no evidence for aberrant expression of P2X7 receptors in P2x4 tdTomato mice was observed: electrophysiological recordings from tdTomato positive neurons did not show any somatic ATP-evoked currents that may indicate aberrant P2X7 expression in relation to wildtype mice and the P2X7 receptor agonist BzATP did not increase mIPSC frequency onto POMC neurons.
Hemizygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. As of October 2017, it is the experience of The Jackson Laboratory that homozygous mice are viable and fertile.

Development

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.

Allele

Symbol: Tg(P2rx4-tdTomato)1Khakh
Name: transgene insertion 1, Baljit Khakh
MGI accession ID: MGI:5779918
Generation method: Transgenic
Attributes: Reporter
Marker symbol: Tg(P2rx4-tdTomato)1Khakh
Marker name: transgene insertion 1, Baljit Khakh
Chromosome: UN
Strain of origin: (C57BL/6 x DBA/2)F1
Expressed genes: RFP,Red Fluorescent Protein,coral
Site of expression: These transgenic mice exhibit widespread distribution of tdTomato-expressing cells throughout the brain in a pattern that represents P2rx4 expression.
Molecular note: The ~191 kbp C57BL/6J BAC RP23-448O6A containing the P2rx4 gene was modified by insertion of a tdTomato cDNA sequence with a BGH polyadenylation sequence into the ATG start site of the P2rx4 gene (replacing the translation initiation codon in exon 1).