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L. L. Daniele, R. K. Chance, J. Wang, E. N. Pugh, Jr.; Insights Into Mechanisms Regulating Cone Opsin Expression From S-opsin "Knock-out" Mice. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3246.
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To test the effects of disruption of the mouse Opn1sw (S-opsin) gene on cone photoreceptor function and cone opsin expression.
In the process of creating a "knock-in" mouse with a targeted point mutation a NeoR cassette with a stop codon was introduced into the Opn1sw gene locus at intron 3-4. Oriented retina whole mounts and cryosections were labeled with immunosera to detect Arrestin-4 (mCarr), S-opsin and M-opsin. Cone b-waves were recorded from Opn1swNeo/Neo and WT mice using electroretinography. Single cell recordings of Opn1swNeo/Neo cones from known retinal locations were made to investigate spectral sensitivity. Total RNA was isolated from eyes of Opn1sw Neo/Neo and WT mice and subjected to quantitative real-time RT-PCR (qRT-PCR) analysis to investigate changes in expression of cone opsins.
No S-opsin positive cells were detected in retinas of Opn1swNeo/Neo mice. However cone number and morphology appeared unaltered. Opn1swNeo/Neo mouse cone b-wave sensitivity peaked at 510 nm with a ratio of sensitivity at 360 nm relative to 510 nm (S360/S510) of 0.4. In contrast, WT cone b-wave sensitivity peaks at 360 nm having a secondary peak at 510 nm with S360/S510 = 4.6. Interestingly, absolute cone b-wave sensitivity at 510 nm increased in Opn1swNeo/Neo mice by ~ 2-fold. S-opsin transcript was ~ 5-fold more abundant than M-opsin transcript in WT retinas, consistent with ERG spectral sensitivities. In Opn1swNeo/Neo mice transcript levels of S-opsin (exon 4-5 probe) relative to rhodopsin were reduced by ~1000-fold compared with WT, while M-opsin transcript levels relative to rhodopsin were not significantly different. The spectral sensitivity of single cones of Opn1swNeo/Neo mice from the mid-ventral retina, the sector with the highest expression of S-opsin in WT retinas, peaked at ~510 nm, consistent with expression of M-opsin as the sole visual pigment.
Opn1swNeo/Neo mice express no functional cone S-opsin, yet our data indicate no decrease in the total number of cones. The two-fold increase in ERG b-wave sensitivity at 510 nm suggests a possible increase in the levels of M-opsin. Increased M-opsin in Opn1swNeo/Neo mice suggests a mechanism that operates to conserve a fixed concentration of opsin in cone photoreceptors. Given the negative qRT-PCR results, altered transcription is not a plausible mechanism. Other mechanisms such as increased translation or increased trafficking of M-opsin to outer segments are under investigation.
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