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Fig. 1. Targeted disruption of O/E genes. (A) Schematic of O/E2
targeting strategy. The taulacZ-pA-HSV-tk(x)-pgk-neo cassette was
inserted into the 5' UTR of the O/E2 gene, replacing the first
six exons encoding the first 185 amino acids of the O/E2 protein, and placing
the tau-lacZ-pA cassette under the transcriptional control of the
endogenous O/E2 promoter. The thymidine kinase (tk) cassette
was inactivated by deleting a 476 bp BstBI-NruI fragment to
permit transmission through the male germline. Mice were generated with ES
cells carrying mutation in the O/E2 locus and mated to cre-expressing
mice to remove the HSV-tk(
)-pgk-neo cassette flanked
by loxP sites. The position of EcoRI restriction enzyme recognition
sites and the location of the probe used for Southern blot confirmation of
homologous recombination are indicated. (B) Schematic of O/E3
targeting strategy. The strategy is similar to the O/E2 targeting
strategy with the following changes. A tau-GFP-pA reporter gene was
used in the place of tau-lacZ-pA cassette, and five exons of the
O/E3 gene encoding the first 162 amino acids of the O/E3 protein were
replaced. (C) Southern blot analysis of the O/E2 alleles. Genomic DNA
of O/E2 mutant littermates was digested with EcoRI
restriction enzyme, separated by agarose-gel filtration and subjected to
Southern blot analysis. A wild-type O/E2 allele yields a 8 kb
hybridization signal and a mutated O/E2 allele gives a 6 kb signal.
(D) Southern blot analysis of the O/E3 alleles. A wild-type
O/E3 allele gives a 7.5 kb hybridization signal and a mutated
O/E3 allele gives a 3 kb hybridization signal. (E) In situ
hybridization of olfactory epithelium sections of neonatal O/E2
heterozygous and homozygous animals with O/E1, O/E2 and O/E3 probes. (F) In
situ hybridization of olfactory epithelium sections of neonatal O/E3
heterozygous and homozygous animals with O/E1, O/E2 and O/E3 probes.
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