It has been demonstrated that retinal photoreception among mammals extends beyond rods and cones to include a small number of intrinsically photosensitive retinal ganglion cells (pRGCs), which are capable of responding to light due to expression of the melanopsin (OPN4) photopigment. OPN4 may have therapeutic potential if ectopically expressed in the degenerate retina in cases where photoreceptors are lost, but the other molecules involved in this light induced transduction cascade are less well characterized. Therefore I sought to probe further the mechanism of OPN4 mediated light sensitivity by siRNA mediated knock down of specific molecules in two mice models in which complete loss of rods and cones renders them almost exclusively dependent on the OPN4 pathway for light sensitivity. I generated siRNA probes against the long transcript variant of murine Opn4 mRNA and first tested these probes on the murine Neuro2A (N2a) cell line, before assessing effects in C3H/HeN rd and rodless/coneless rd/rd cl mice. siRNA was injected intravitreally into one eye and pupillometry was assessed, combined with molecular analyses at various timepoints. Reverse transcription polymerase chain reaction (RT-PCR) analysis in N2a cells confirmed Opn4 knockdown and immunolabelling techniques identified >85% silencing with siRNA. Pupil responses in the rd and rd/rd cl mice were inhibited by the siRNA injections in vivo which confirmed the functional effect of Opn4 silencing detected by molecular analysis. I therefore present a novel reproducible in vivo model in which siRNA induced silencing of the melanopsin pathway can be assessed by pupillometry and compared to levels of mRNA and protein at specific timepoints. Probes against other putative candidate genes, such as TRPC3, may unravel the molecular interactions of this pathway. This may help in future to induce light sensitivity in other retinal neurons in patients who are completely blind from photoreceptor loss.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:627770 |
| Date | January 2012 |
| Creators | Vachtsevanos, Athanasios |
| Contributors | MacLaren, Robert E. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:631deeeb-90c0-4e90-b24e-f03e1b318d8b |
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