Molecular dissection of the coneless transgenic mouse retina
Freedman MS., Von Schantz M., Soni BC., Foster RG.
Purpose. In addition to their role in vision, cone cells are strong candidates for mediating the arcadian responses to light in mammals. Using confess transgenic (Cl) mice (generously donated by J. Nathans and Y. Wang, Johns Hopkins University) consisting of 6.2kb of the human red cone promoter, linked to the coding region of an attenuated diphtheria toxin gene, we have begun a molecular characterization of the retina in order to determine the extent of this genetic lesion, in animals of 45 days of age. Methods. We used Reverse Transcriptase-Polymerase Chain Reaction (RTPCR) to amplify cone-specific messages from retinal cDNA. Various sets of primers designed to amplify cone-specific transcripts were used for PCR amplification. Reanlta. RT-PCR using primers specific for either green cone or UV cone opsins resulted in a product only for the latter, whereas both messages were amplified in the wild type. Further investigation indicates no presence of C-arrestin or a putative green cone-specific gamma phosphodiesterase in the Cl retina. Alpha-2-transduän and alpha phosphodiesterase, which are found in all cone types, were amplified in both wild type and Cl cDNA. Subsequent cloning and sequencing of these products will confirm their identity. Conclusions. No messages specific for green cones could be detected in cDNA from the CI retina. Messages expressed in UV cones were present, although at reduced levels. We intend to extend the analysis to include animals of different ages. Future work will also involve analyzing arcadian responses to light of these animals, and we will correlate this molecular study with the results of the behavioural investigation in order to elucidate exactly how these mammals regulate their biological clock.