B6;129P2-Omp^tm2(spH)Mom/J

Stock number: 004946
Product name: OMP-spH KO
Strain synonyms: OMP-spH mice, OMP-synapto-pHluorin mice
Research areas: Neurobiology Research, Research Tools

Description

These Omp knock-out, reporter mice express synapto-pHluorin (spH) fluorescence in all glomeruli of the olfactory bulb.

Detailed description

These mutant mice express synapto-pHluorin (spH) from the endogenous locus encoding the olfactory marker protein (OMP) as a result of a targeted knockin mutation that replaces the OMP coding region with that of spH. The OMP locus directs expression of spH at high levels and exclusively in mature sensory neurons of the main olfactory epithelium (OSNs) and the vomeronasal epithelium. SpH is a pH-sensitive variant of the green fluorescent protein (ecliptic pHluorin) fused to the mouse synaptic vesicle-associated protein (VAMP2). The protein is localized preferentially in synaptic vesicles, but is also present on the plasma membrane of OSN axons and nerve terminals. The fluorescent domain of spH is exposed to the acidic lumen of synaptic vesicles where it is ~ 20 fold less fluorescent than at neutral pH. Upon synaptic release, the lumen of the synaptic vesicles becomes continuous with the extracellular space resulting in an increase in pH that causes a rapid increase in fluorescence. In mutant mice all glomeruli of the olfactory bulb are robustly labeled. There are ~1800 glomeruli in the main olfactory bulb, and each glomerulus receives axonal input from thousands of OSNs that express the same odorant receptor. Glomeruli in the dorsal surface of the olfactory bulb can be observed in vivo, after thinning the skull, through a standard epifluorescence microscope. Fluorescence changes caused by neuronal activity and presynaptic release can be measured in living mice following odorant stimulation. Mice that are homozygous for this allele do not express gene product (OMP) but are viable, fertile, normal in size and do not display gross physical or behavioral abnormalities. Homozygous mice produce small litters and breed at reduced frequency. This mutant mouse strain should prove useful to measure stimulus-induced activity or presynaptic vesicle release in OSNs of living mice.

Development

A targeting vector containing super-ecliptic synapto-pHluorin coding sequence and an auto-excising neomycin resistance cassette was used to replace the Omp coding region sequence in 129P2/OlaHsd-derived E14 embryonic stem (ES) cells. The loxP-flanked auto-excising neomycin resistance cassette is composed of the mouse angiotensin-converting enzyme promoter (tACE) located 5' of sequences encoding Cre recombinase and the neomycin resistance gene. The angiotensin-converting enzyme promoter has been shown to initiate transcription only during spermatogenesis. Correctly targeted ES cells were injected into C57BL/6 blastocysts and a chimeric animal derived from cell clone OSE-80 was obtained.

The plasmid that was used to generate this mutation is banked and described at Addgene.

Allele

Symbol: Omp^tm2(spH)Mom
Name: targeted mutation 2, Peter Mombaerts
MGI accession ID: MGI:2684190
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Omp
Marker name: olfactory marker protein
Chromosome: 7
Strain of origin: 129P2/OlaHsd
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: active mature sensory neurons of the main olfactory epithelium (OSNs) and the vomeronasal epithelium; protein localized preferentially in synaptic vesicles, but is also present on the plasma membrane of OSN axons and nerve terminals; all glomeruli of the olfactory bulb are robustly labeled (visible in vivo); pH sensitive
Molecular note: The coding exon was replaced by a construct containing the ecliptic synaptopHluorin (spH) coding sequence and a self-excising neomycin resistance cassette. SpH is a fusion protein joining a pH-sensitive derivative of wild-type green fluorescent protein (GFP) to the carboxyl terminus of a mouse synaptic vesicle-associated membrane protein (VAMP2), which targets spH to the lumenal face of pre-synaptic vesicles. The loxP-flanked, male-germline self-excising neomycin resistance cassette (which was deleted in the chimeric founder's germline) contains the Cre recombinase coding sequence under control of the mouse testis-specific angiotensin-converting enzyme (tACE) promoter and tk-neo. The endogenous gene regulatory sequences direct spH/Vamp expression specifically to sensory neurons in glomeruli of the olfactory bulb.
General note: In the acidic lumen of pre-synaptic vesicles, the fluorescence of spH is only about 5% of its intensity at neutral pH. When synaptic vesicles with spH anchored to their lumenal face fuse with the plasma membrane to release their contents, the spH is exposed to the pH-neutral extracellular environmment and rapidly becomes highly fluorescent. All olfactory bulb glomeruli fluoresce, with varying intensity. The superficial nerve layer overlying the glomerular layer and comprising axons of olfactory sensory neurons is more weakly fluorescent. In vivo fluorescent imaging of the olfactory bulb demonstrates that odorant-evoked responses cause strong fluorescence of odorant-specific, bilaterally symmetric groups of glomeruli.