Overview

Also Known As: P2rx4 tdTomato BAC transgenic

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.

Donating Investigator

Baljit S Khakh, University of California Los Angeles

Genetic overview

Genetic Background	Generation

Tg(P2rx4-tdTomato)1Khakh

Allele Type
Transgenic (Reporter)

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Research Applications

Research Tools
Neurobiology Research

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Base Price

Starting at:

$2,854.50 Domestic price Cryo Recovery

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Details

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.

Expression Data

Expressed Gene	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.

Control Suggestions

Approximate Controls

005304 C57BL/6NJ

Additional Information

Considerations for Choosing Controls

Genetics

Tg(P2rx4-tdTomato)1Khakh

Allele Symbol: Tg(P2rx4-tdTomato)1Khakh

Allele Name	transgene insertion 1, Baljit Khakh
Allele Type	Transgenic (Reporter)
Allele Synonym(s)	Tg(P2rx4-tdTomato)1Khakh; transgene insertion 1, Baljit Khakh
Gene Symbol and Name	Tg(P2rx4-tdTomato)1Khakh, transgene insertion 1, Baljit Khakh
Gene Synonym(s)
Promoter	P2rx4, purinergic receptor P2X, ligand-gated ion channel 4, mouse, laboratory
Expressed Gene	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.
Strain of Origin	(C57BL/6 x DBA/2)F1
Chromosome	UN
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).

Disease/Phenotype

Disease Terms

Research Areas By Genotype

This mouse can be used to support research in many areas including:

Research Tools
- Fluorescent Proteins
- Genetics Research
  - Tissue/Cell Markers: neurons
- Neurobiology Research
  - cell marker
Neurobiology Research
- Fluorescent protein expression in neural tissue

Mammalian Phenotype Terms by Genotype

References

Additional - Tg(P2rx4-tdTomato)1Khakh related

Technical Support

Contact Technical Support

Genotyping Protocols
- Separated PCR: Tg(P2rx4-tdTomato)1Khakh-Alternate 2
- Genotyping resources and troubleshooting
Breeding Considerations

Hemizygous mice are reported to be viable and fertile. As of October 2017, it is the experience of The Jackson Laboratory that homozygous mice are viable and fertile, and our live colony is maintained by breeding homozygous mice together.
- Additional Breeding and Husbandry Support
Citation
When using the P2rx4 tdTomato BAC transgenic mouse strain in a publication, please cite the originating article(s) and include JAX stock #029332 in your Materials and Methods section.

Animal Health Reports

Facility Barrier Level Descriptions

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Pricing & Availability

Cryo Recovery

Domestic
International

Live Mouse

Age

Genotype

Price

weeks

Cryorecovery - Pricing

Service	Genotype	Price
	Hemizygous or Non carrier or Tg(P2rx4-tdTomato)1Khakh

We will fulfill your order by providing at least two carriers for each strain ordered. The total number, sex, and genotypes provided will vary, although typically 8 or more animals are provided. Please check genotypes which will be recovered. While the genotypes of all animals produced will be communicated to you prior to scheduling shipment, the genotypes of animals provided may not reflect the mating scheme and genotypes described in the strain description. Animals are typically ready to ship in 11-14 weeks. If a second recovery is required to produce the minimum number of animals, then delivery time would increase to approximately 25 weeks. If we fail to produce animals of the correct genotype, you will not be charged. We cannot guarantee the reproductive success of mice shipped to your facility. If the mice are lost after the first three days (post-arrival) or do not produce progeny at your facility, a new order and fee will be necessary.

Cryorecovery to establish a Dedicated Supply for greater quantities of mice. Mice recovered can be used to establish a dedicated colony to contractually supply you mice according to your requirements. Price by quotation.

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Also Known As: P2rx4 tdTomato BAC transgenic

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Approximate Controls

Additional Information

Tg(P2rx4-tdTomato)1Khakh

Allele Symbol: Tg(P2rx4-tdTomato)1Khakh

Disease Terms

Research Areas By Genotype

This mouse can be used to support research in many areas including:

Mammalian Phenotype Terms by Genotype

References

Additional - Tg(P2rx4-tdTomato)1Khakh related

Genotyping Protocols

Breeding Considerations

Citation

Animal Health Reports

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Live Mouse

Cryorecovery - Pricing

Related Products and Services

Payment Terms and Conditions

The Jackson Laboratory's Genotype Promise

Terms of Use

Additional Use Restrictions Apply

Licensing Information

JAX® Mice, Products & Services Conditions of Use

No Warranty

Credit for PRODUCTS or SERVICES

Animal Care and Use for SERVICES

No Liability