B6.Cg-Pex5l^tm1.1Sieg/J

Stock number: 038046
Product name: TRIP8b^flox
Strain synonyms: Pex5l^flox
Research areas: Cardiovascular Research, Neurobiology Research, Research Tools

Description

TRIP8b^flox mice have loxP sites flanking exon 7 of the peroxisomal biogenesis factor 5-like (Pex5l) gene. These mice may be useful for studying the role of hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels in neuronal responses.

Detailed description

TRIP8b^flox mice have loxP sites flanking exon 7 of the peroxisomal biogenesis factor 5-like (Pex5l) gene. TRIP8b is an auxiliary (beta) subunit for the hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel family (HCN1-4), members of which are expressed in heart and brain cells where they are involved in the generation and modulation of cardiac rhythmicity and neuronal responses to synaptic input. Specifically, TRIP8b is required for HCN channel expression in excitatory neurons in the brain and spinal cord and is necessary for dendritic expression of HCN1.

TRIP8b^flox homozygous mice are viable and fertile. When bred to mice that express tissue-specific Cre recombinase, resulting offspring will have coding exon 7 deleted in the Cre-expressing tissues. Deletion of exon 7 leads to a frameshift mutation in the coding sequence with the formation of an unstable TRIP8b protein, undetectable by Western blot analysis using N-terminal antibodies.

When bred to CMV-cre mice (Stock No. 006054) with widespread Cre expression, resulting TRIP8b^-/- mice have strongly reduced levels of HCN1, HCN2 and HCN4 protein in the brain, which leads to a variety of functional consequences in neurons such as absence seizures and various behavioral abnormalities.

Development

The L1L2_Bact_P cassette was inserted at a position on Chromosome 3 upstream of coding exon 7 of the peroxisomal biogenesis factor 5-like (Pex5l) gene. The cassette is composed of a FRT site, an internal ribosome entry site (IRES), a β-galactosidase (lacZ) sequence, a loxP site, and a neomycin resistance (neo) cassette, followed by a second FRT site and a second loxP site. A third loxP site was inserted downstream of coding exon 7, according to the Ensembl Canonical reference Transcript: ENSMUST00000194016.6 Pex5l-211. This exon is referenced as exon 8 in some references, including Srinivas et al. 2017 J Neurosci. 22;37(12):3276-3293 (PMID: 28213444), due to the variability in splice variants, some of which include an "exon 3" noted by the donating laboratory of Dr. Steven Siegelbaum (Columbia University).

Dr. Siegelbaum obtained this Pex5l^{tm1a(EUCOMM)Wtsi} targeting vector (PRPGS00115_B_D04) and electroporated it into (C57BL/6J x 129S6/SvEvTac)F1-derived KV1 embryonic stem (ES) cell. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric mice were bred to ACTB:FLPe mice (Stock No. 005703) to remove the FRT-flanked region containing the lacZ and neo cassette sequences. Resulting TRIP8b^flox mice were bred to C57BL/6J mice (Stock No. 000664) to remove the Flp recombinase and mice were further bred to C57BL/6J mice for at least 8 generations. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

Allele

Symbol: Pex5l^tm1.1Sieg
Name: targeted mutation 1.1, Steven A Siegelbaum
MGI accession ID: MGI:6342268
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed)
Synonyms: TRIP8b^flox
Marker symbol: Pex5l
Marker name: peroxisomal biogenesis factor 5-like
Marker synonyms: 1700016J08Rik; Pex2; PEX5R; PEX5RP; PXR2; PXR2B; TRIP8b; Trjip8b
Chromosome: 3
Strain of origin: (C57BL/6J x 129S6/SvEvTac)F1
Molecular note: Using vector PRPGS00115_B_D04 from IMPC, the cassette inserted at position 33004820 of Chromosome 3 upstream of the critical exon 7 (Build GRCm38). The cassette is composed of an FRT site followed by lacZ sequence and a loxP site. This first loxP site is followed by a neomycin resistance gene under the control of the human beta-actin promoter, SV40 polyA, a second FRT site and a second loxP site. A third loxP site is inserted downstream of the targeted exon at position 33004060. The critical exon(s) is/are thus flanked by loxP sites. Flp-mediated recombination removed the reporter and selection cassette.