129S6.129P2(B6)-Gucy1b2^tm2Mom/MomJ

Stock number: 021844
Product name: 129S6-Gucy1b2-KO
Research areas: Cell Biology Research, Neurobiology Research, Research Tools, Sensorineural Research

Description

Type B olfactory sensory neurons have been shown to respond rapidly and in a graded manner to reduced environmental oxygen levels. Type B cells from mice homozygous for this targeted null allele fail to flux calcium in response to reduced oxygen and homozygotes fail to respond normally to a low oxygen environment in a conditioned place aversion test.

Detailed description

Type B olfactory sensory neurons of the main olfactory epithelium have a distinctive expression profile, most notably expressing GUCY1B2, CNGA2, and TRPC2 (a cation channel typically expressed by vomeronasal sensory neurons), but not expressing ADCY3 or odorant receptors, which usually are expressed by olfactory sensory neurons. Type B cells have been shown to respond rapidly and in a graded manner to reduced oxygen levels and this response requires functional GUCY1B2 and TRPC2, as well as extracellular calcium cations. Type B cells from wild type mice flux calcium in response to 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, and 8-bromo-cGMP, but were found not to respond to forskolin, carbon disulfide, mouse urine, or an array of other odorants. Inhibition of nitric oxide synthases did not interfere with the response of type B cells to reduced oxygen. Strong expression of Gucy1b2 in the brain appears restricted to this subset of olfactory sensory neurons.

Type B cells from mice homozygous for this targeted null allele of Gucy1b2 respond normally to 8-bromo-cGMP or KCl, but fail to flux calcium in response to reduced oxygen. The impact of this was evidenced by assessing the activation of postsynaptic olfactory bulb neurons in the vicinity of type B cell glomeruli subsequent to exposure to environmental hypoxia. These post-synaptic olfactory bulb neurons showed increased FOS immunoreactivity, indicative of activation, in controls and heterozygotes but not in homozygotes. The essential role for GUCY1B2 in environmental hypoxia sensing was further confirmed in a conditioned place aversion test. Wild-type controls and heterozygotes responded with place aversion to a reduced oxygen area, where homozygotes did not. Homozygotes on the 129S6 congenic background were found to have a normal hyperventilation response to low environmental oxygen, consistent with normal carotid body function, further proving that Type B cells assess the oxygen content of the air, not the oxygenation in the body. Homozygotes performed normally in a Morris water maze for both latency to reach the platform and reference memory.

Development

This targeted mutation has a deletion of approximately 4.3 kb including part of exon 12 through part of exon 15, which encompasses the catalytic domain. Additionally a stop codon, AscI site, and an IRES-tauGFP-pA ANCF cassette were inserted adjacent to the deletion in exon 12 resulting in a null allele with GFP reporter expression in place of the endogenous protein. This construct was injected into 129P2/OlaHsd-derived E14 ES cells and the resulting chimeras were bred to C57BL/6J. This strain was maintained by sibling inbreeding on a segregating background and subsequently backcrossed to 129S6/SvEvTac for 10 generations, and sperm was cryopreserved from congenic heterozygous males at generation N10.

Allele

Symbol: Gucy1b2^tm2Mom
Name: targeted mutation 2, Peter Mombaerts
MGI accession ID: MGI:5444154
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Gucy1b2
Marker name: guanylate cyclase 1, soluble, beta 2
Chromosome: 14
Strain of origin: 129P2/OlaHsd
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: Gucyb1b2 is expressed in type B chemosensory neurons of the main olfactory epithelium. Because the GFP sequence is spliced out of the mutant transcripts, the cells expressing the mutant allele do not express GFP.
Molecular note: Approximately 4.3 kb of DNA encoding the catalytic domain, spanning from partway through exon 12 to partway through of exon 15, was deleted, a stop codon and AscI site was generated in the middle of exon 12, and an IRES-tauGFP-pA ACNF cassette was inserted into the AscI site thus creating a null allele with GFP reporter.