Gene Delivery using Adenoviral Vector into the Organotypic Culture System of the Chick Retinae
Keywords:
Gene delivery, Adenoviral vector, Retina, Tissue culture, Green Fluorescent Protein (GFP)Abstract
Damage to retina during development correlates with many ophthalmological pathologies, such as retinal dysplasia and degeneration. Virus-mediated gene transfer into identified retinal cells may offer a great potential for therapeutic interventions. This study describes a three-dimensional culture system of the embryonic chick retina. Samples from 17 and 19 days old retinae were isolated from eyeball and were cultured for up to 10 days. Selection of embryonic stage and specimen processing were optimized for culturing. The ability of a replication-deficient adenovirus vector (AVV) carrying an early cytomegalovirus (CMV) gene promoter to efficiently transfer a green fluorescent protein (GFP) gene into the cultured embryonic chick retinae was analysed. The results indicated that retinal tissue slices did not show any change in their cytoarchitecture. The results also showed that the GFP expression was maximal about 24 hours after infection of retinal slices and were maintained for several days. They also showed that GFP expression was widespread in the retinal slices. Moreover, the results also showed that injecting of AVV did not show any detectable cytopathic effects - these were only observed for very high titres of infection. The approach developed here is a valuable tool that can be used to assess the effect of pharmacological manipulations on retinal cells and to transfer of effective therapeutic genes into the retina, which might also facilitate the search for understanding the mechanisms
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References
Clark, A.F. and T. Yorio, Ophthalmic drug discovery. Nature Review 2003. 2: p. 448–459.
2. Kato, S., Negishi, K., Hayashi, Y., Miki, N., Enhancement of neurite outgrowth and aspartate-glutamate uptake systems in retinal explants cultured with chick gizzard extract. Journal of Neurochemistry, 1983. 40: p. 929-938.
3. Hoff, A., Hämmerle, H., Schlosshauer, B., Organotypic culture system of chicken retina. Brain Research Brain Research, 1999. 4: p. 237-248.
4. Rzeczinski, S., Victorov, I.V., Lyjin, A.A., Aleksandrova, O.P., Harms, C., Kronenberg, G., Freyer, D., Scheibe, F., Priller, J., Endres, M., Dirnagl, U., Roller culture of free-floating retinal slices: a new system of organotypic cultures of adult rat retina. Ophthalamic Research, 2006. 38: p. 263-269.
5. Caffé, A.R., Viser, H., Jansen, H.G., Sanyal, S., Histotypic differentiation of neonatal mouse retina in organ culture. Current Eye Research, 1989. 8: p. 1083-1092.
6. Johnson, T.V., Martin, K.R., Development and characterisation of an adult retinal explant organotypic tissue culture system as an in vitro intraocular stem cell transplantation model. Invest. Ophthalmol. Vis. Sci. , 2008. 49: p. 3503-3512.
7. Melani, A., et al., Selective adenosine A2a receptor antagonism reduces JNK activation in oligodendrocytes after cerebral ischaemia. Brain, 2009. 132: p. 1480-1495.
8. Stoppini, L., P.-A. Buchs, and D. Muller, A simple method for organotypic cultures of nervous tissue. Journal of Neuroscience Methods, 1991. 37: p. 173-182.
9. Gähwiler, B.H., et al., Organotypic slice cultures: a technique has come of age. Trends Neuroscience, 1997. 20: p. 471-477.
10. Stokes, C.E.L., et al., Dynamics of a transgene expression in acute rat brain slices transfected with adenoviral vectors. Experimental Physiology, 2003. 88: p. 459-466.
11. Kluge, A., et al., Tracing of the entorhinal-hippocampal pathway in vitro. Hippocampus, 1998. 8: p. 57-68.
