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Gabriel Peinado, Cecilia Gerstner, Wolfgang Baehr, Edward Pugh; Targeted deletion of the X-linked mouse cone M-opsin gene reveals competition between M- and S-opsin expression. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1334.
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© ARVO (1962-2015); The Authors (2016-present)
To assess the consequences of targeted deletion of Opn1mw.
The targeting construct inserted a gene trap into intron 2 of the Opn1mw gene. The trap comprises a splice-acceptor sequence upstream of a promoterless β-geo reporter, lacZ and neomycin selection cassette. Successful targeting was confirmed by PCR analysis. Wild type, heterozygotes and knockout (KO) litter mates between 2 and 3 months of age were maintained on a 12D:12L light cycle, dark adapted overnight and anesthetized with 1.5% isofluorane. ERGs were measured with a Phoenix Labs Maxwellian view system using 1 ms 365 nm and 505 nm LED stimulation. Cone-driven ERG b-waves were recorded in the presence of a 505 nm background that suppressed the rod a-wave. B-wave response (R) vs. intensity (I) data were fitted with exponential saturation functions to extract the saturating amplitude Rmax, and sensitivity (S365, S505) to the 365 nm and 505 nm stimuli. Western blot analysis was performed with cone opsin antibodies.
PCR analysis and Western blotting confirmed that M-opsin expression was greatly reduced in male mice with the targeted allele. The average saturating rod a-wave amplitude was 950 μV; cone b-wave amplitude Rmax was 180 μV. Three distinct results were observed in the cone b-wave data. (1) S505 was reduced more than 1000-fold in mice whose X-chromosome(s) express(es) the gene trap, supporting their KO status. (2) S365 was about 2-fold higher for KO than for WT mice. (3) R vs I data of heterozygous females for 505 nm light exhibited two “branches”: the more sensitive branch had S505 about the same as WT males and saturated around 6000 photons mm-2, while S505 for the less sensitive branch was close to that of KO mice.
Most mouse retinal cones co-express S- and M-opsin, in complementary dorso-ventral gradients. Deletion of S-opsin results in an increased expression of M-opsin (Daniele et al., 2011), hypothesized to arise from elimination of competition of M-opsin mRNA with that of S-opsin for ribosomes in cones that would normally co-express both cone opsins. Result (2) provides further support this hypothesis, as elimination of M-opsin mRNA would allow normally co-expressing cones to produce more S-opsin. Result (3) may arise from X-inactivation, as in females heterozygous for the targeted allele many cones would be expected to have their “good” Opn1mw allele inactivated.
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