B6;129P2-Or8a1^{tm35(Adrb2)Mom}/MomJ

Stock number: 006734
Research areas: Developmental Biology Research, Neurobiology Research, Research Tools, Sensorineural Research

Description

This targeted substitution of the beta 2 adrenergic receptor in place of olfactory receptor family 8 subfamily a member 1 is valuable for the study of olfaction, and olfactory sensory neuron signaling, function, and development.

Detailed description

This targeted substitution of the beta 2 adrenergic receptor (Adbr2) in place of olfactory receptor 8a1 (Or8a1 formerly Olfr151 or M71) drives expression of this non-olfactory G-protein coupled receptor in a pattern consistent with normal Or8a1 expression as assessed by the bicistronically expressed tauGFP marker. At three weeks of age an average of 1,262 cells in the dorsal main olfactory epithelium express GFP in homozygotes. Co-staining for OMP and GAP43 shows that these olfactory sensory neurons are, as a population, somewhat less mature than wild-type Or8a1-expressing olfactory sensory neurons. These Adbr2-expressing olfactory sensory neurons respond in a dose-dependent manner to isoproterenol, but not to acetophenone, a ligand for Or8a1. Wholemount immunohistochemistry shows that Adbr2 protein localizes in the dendritic cilia of the olfactory sensory neurons, similar to localization of olfactory receptor proteins. When crossed with Or8a1^tm26Mom homozygotes, which have an IRES-taulacZ insertion bicistronically expressed downstream of the endogenous Or8a1 coding sequence (see stock 006676) the compound heterozygous offspring show no double-positive cells. This monoallelic expression proves that allelic exclusion of olfactory receptors does not require the endogenous olfactory receptor protein or DNA sequence. Expression of olfactory receptors and Adbr2 in the main olfactory epithelium, assessed by in situ hybridization, strongly suggests that olfactory sensory neurons that express Adbr2 do not co-express an olfactory receptor, so this substitution permits Adbr2 to function in place of an olfactory receptor with normal monogenic expression and allelic exclusion. In heterozygotes the axons of these Adbr2-expressing olfactory sensory neurons coalesce into homogeneous glomeruli distinct from M71 glomeruli. While there are approximately the same number of M71 glomeruli and Adbr2 glomeruli, the Adbr2 glomeruli are smaller, by approximately half. In compound heterozygotes expressing this allele and Or8a1^{tm14(Adrb2)Mom}, which has the same Adbr2 substitution but with a taulacZ bicistronic marker (see stock 006691), the Adbr2 expressing axons coalesce into the same glomeruli.

Development

The full coding sequence Or8a1 (formerly Olfr151) was excised and replaced with the full coding sequence of beta 2 adrenergic receptor, followed by an IRES-tauGFP-ANCF cassette placed 3 bases downstream of the stop codon. This targeting vector was electroporated into 129P2/OlaHsd-derived E14 ES cells in the laboratory of Dr. Peter Mombaerts. Male chimeras were bred to C57BL/6J females and the self-excising neomycin resistance sequence in the ACNf cassette was removed during transmission through the male germline. This segregating B6;129P2 line was maintained by sibling intercrossing and sperm was cryopreserved from homozygous males.

Allele

Symbol: Or8a1^{tm35(Adrb2)Mom}
Name: targeted mutation 35, Peter Mombaerts
MGI accession ID: MGI:3722388
Generation method: Targeted
Attributes: Reporter; Inserted expressed sequence
Marker symbol: Or8a1
Marker name: olfactory receptor family 8 subfamily A member 1
Chromosome: 9
Strain of origin: 129P2/OlaHsd
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: GFP is expressed in the dorsal main olfactory epithelium.
Molecular note: The endogenous coding sequence for this olfactory receptor was replaced with the coding sequence for beta 2 adrenergic receptor with an IRES-tauGFP-ANCF cassette inserted 3 nucleotides after the stop codon to yield bicistronic expression of beta 2 adrenergic receptor and GFP in place of this olfactory receptor. The ACNf neomycin selection cassette self-excised during transmission through the male germline leaving a single loxP site.