| Research Abstract |
In this project, we attempted to express exogeneous genes and knock down endogeneous genes with antisence oligonucleotides in newt retinas. Our results can be summarized as follows.
An expression vector of EGFP gene under the control of a CMV promotor was introduced with cation liposome in viteras of adult newt eyes. After three days from the operation, retinal sections were examined with a fluorescent microscope, but no fluorescence from EGFP was detected. Then we attempted to detect the EGFP by immunohistochemical investigation using anti-GFP antibodies. Antibodies were recognized ganglyon cell layer, inner plexiform layer and inner nuclear layer of the retinas suggesting that EGFP genes were expressed in the newt retinas. However; the amount of EGFP expressed in the retina is likely limited. So, we are now investigating the best condition for gene expression such as expression vectors, lipofection reagents, and the amount of introduced vectors.
In order to knock down a gene expression
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with anti-sense oligonucleotides, accurate nucleotide sequence of each newt gene should be clarified. We isolated mRNAs from adult normal newt retinas and regenerating newt retinas after 18-19 days from the surgical removal of normal retinas. Two kinds of cDNA libraries, normal and regenerating retinal cDNA libraries, were constructed. Several hundred clones were randomly isolated from these libraries, and nucleotide sequences were determined. Homology of these sequences to the sequences deposited in the databases, and function of each gene was elucidated. We found cDNAs encoding four kinds of opsins (protein moieties of visual pigments), OTX2 (a homeodomain protein responcible retinal development), and stathmin (a protein responcible for axonal regeneration). Expression of these genes were investigated by in situ hybridization. It is clarified that rod cells begin to express opsin before outer segments were formed, and that otx2 and stathmin genes are strongly expressed in regenerating newt retinas. Less
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