Abstract
Purpose: :
To create a "knock-in" mouse that expresses S-cone opsin with a max shifted from 360 nm to ~ 430 nm.
Methods: :
A single point mutation was introduced into mouse Opn1sw (S-opsin) through gene targeting resulting in mice expressing S-opsin with a Phenylalanine to Tyrosine replacement at residue 81 (F81Y). Cone function and spectral sensitivity was assessed in mutant and WT mice by measuring cone b-wave responses (electroretinography) or single cone responses (suction pipette recordings) elicited by calibrated light flashes varying in wavelength and intensity.
Results: :
The presence of the targeted Opn1sw gene was confirmed with Southern blotting of BamH1 and Bgl1 restriction fragments of mouse genomic DNA with probes 3’ and 5’ to the targeting vector. The presence of the point mutation in Opn1sw was confirmed using PCR amplification of genomic DNA followed by DNA sequencing. Cone number and morphology appeared unaltered in retinas of F81Y mice. Data from single cell recordings confirm that similar to WT mice the ventral retina of the F81Y mouse is populated with S-dominant cones. The sensitivity of S-dominant cones of F81Y mice peaked at ~ 420 nm light in contrast to a peak at 360 nm for WT cones. The ratio of sensitivity measured at 360 nm to that measured at 420 nm was ~ 0.3-0.4 for F81Y cones and ~ 11 for WT cones. The spectral sensitivity data show a secondary peak in sensitivity at ~510 nm indicating co-expression of M-pigment in S-dominant cones of both WT and F81Y mice. The shape of the spectral sensitivity data from F81Y mouse cones can be reliably described by superposition of Lamb spectral templates for 420 nm and 508 nm, in contrast to WT cone data which are described by Lamb templates for 361 nm and 508 nm. The red shift in the spectral sensitivity of S-opsin (F81Y) was also confirmed from cone b-wave responses.
Conclusions: :
Electrophysiological measurements confirm the shift in peak sensitivity of S-opsin (F81Y) from 360 nm to 420 nm.
Keywords: opsins • photoreceptors • transgenics/knock-outs