B6.Cg-Pikfyve^tm1.1Ashi/J

Stock number: 029331
Product name: PIKfyve^flox (PIKfyve^fl)
Research areas: Cancer Research, Diabetes and Obesity Research, Neurobiology Research, Research Tools

Description

 

The PIKfyve^flox floxed allele has loxP sites flanking Pikfyve exon 6 (encoding the FYVE finger domain). Removal of the floxed sequence creates a null allele. These mice may be useful for generating tissue-specific PIKfyve-deletion for studying phosphoinositides in metabolism, diabetes, cancer, neurological disorders and other human disease.

Detailed description

The enzymes catalyzing synthesis and turnover of phosphoinositides (PI) have been increasing implicated in human diseases, such as diabetes, cancer, neurological disorders and others. While most mammalian PI-metabolizing enzymes have functional redundancies, the phosphoInositide kinase for position five containing a fyve domain gene (Pikfyve) is a product of a single gene, and PIKfyve is responsible for synthesis of nearly the entire cellular amounts for two of the seven PIs (PtdIns5P and PtdIns(3,5)P2). In addition, PIKfyve is a downstream effector of Vps34, a key endosomal trafficking regulator.

The PIKfyve^flox allele (also called PIKfyve^flΔneo) has loxP sites flanking Pikfyve exon 6 (encoding the FYVE finger domain). Mice homozygous for the floxed allele (PIKfyve^flox/flox) are viable and fertile with no reported abnormalities. Upon exposure to Cre recombinase, the deletion of exon 6 causes a frameshift and early stop codon at residue 171 - resulting in no mRNA or functional PIKfyve protein expression (the first 159 amino acid fragment that could have appeared from PIKfyve exons 1-5 is not detected with the PIKfyve N-terminal antibodies; indicating that if synthesized, it is degraded).

When PIKfyve^flox are bred to germline Cre-expressing mice, the resulting PIKfyve global knockout homozygotes die in utero during the preimplantation developmental stage.
After breeding PIKfyve^flox mice with aP2-Cre transgenic mice (Stock No. 005069), the resulting adipose-specific PIKfyve disruption causes severe dysregulation of glucose homeostasis and systemic insulin sensitivity. In addition, those females have severely impaired pregnancy-induced mammary gland differentiation and lactogenesis.
When PIKfyve^flox are bred to have Cre recombinase driven by a muscle creatine kinase promoter (e.g., Mck-Cre [Stock No. 006475]), the resulting animals may be useful in studying systemic glucose homeostasis and insulin-regulated glucose uptake/GLUT4 translocation in skeletal muscle.

Development

The PIKfyve^flox mice were created for Dr. Assia Shisheva (Wayne State University School of Medicine). A targeting vector was designed to insert a loxP site in intron 5, and a frt-flanked PGK-neo cassette followed by a second loxP site just downstream of exon 6 (encoding the FYVE finger domain) of the phosphoinositide kinase, FYVE finger containing locus (Pikfyve) on chromosome 1. The construct was electroporated into B6.Cg-Thy1^a-derived Bruce4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and a chimeric male was bred to C57BL/6 females for germline transmission. Offspring were bred with FlpE-deleter mice (on a C57BL/6J genetic background) for germline deletion of the PGK-neo cassette, resulting in mice with the PIKfyve^flΔneo allele (also called PIKfyve^flox). The PIKfyve^flox colony was bred with C57BL/6 wildtype mice for seven generations (and the FlpE-deleter allele was removed) prior to sending males with black coat color to The Jackson Laboratory Repository in 2011. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm may be used to fertilize C57BL/6J oocytes (Stock No. 000664).
Of note, it is not known if the Y chromosome has been fixed to the C57BL/6J background during backcrossing.

Allele

Symbol: Pikfyve^tm1.1Ashi
Name: targeted mutation 1.1, Assia Shisheva
MGI accession ID: MGI:4949700
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Pikfyve
Marker name: phosphoinositide kinase, FYVE type zinc finger containing
Chromosome: 1
Strain of origin: B6.Cg-Thy1^a
Molecular note: A loxP site was inserted upstream of exon 6. An FRT-flanked neo cassette with a 3' loxP site was inserted downstream of exon 6. Flp-mediated recombination removed the neo cassette.