| Title:
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Modified Ca2+- phosphate method for efficient and rapid transfection of adult rodent whole-mount retinal culture
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| Author(s):
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Leila Azizzadeh, Narsis Daftarian, Shahin Ahmadian, Hamid Ahmadieh, Moghgan Rezaie Kanavi, Mahshid Shafiezadeh
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| Presentation Type:
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Poster
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| Subject:
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Retina and Retinal Cell Biology
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| Others:
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Presenting Author:
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| Name:
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Leila Azizzadeh
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| Affiliation :(optional)
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Institute of Biochemistry and Biophysics IBB, Tehran university
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| E mail:
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leila_azizzadeh@ut.ac.ir
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| Phone:
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Mobile: |
09113475816
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Abstract (Max 200 words)
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| Purpose:
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Primary cultures and transfection are useful tools in determining gene function among specific tissue and developmental stages. Various DNA based constructs have been developed to over expression or knockdown genes of interest and can be delivered into the cells using virus based methods, chemical methods such as cationic lipid, cationic polymer and calcium phosphate and physical methods such as electroporation and biolistic.
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| Methods:
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In this research, we developed a method to culture and transfect of adult rodent retinal cells in order to transfect the post mitotic neuronal cells. In present study, we applied modified calcium phosphate method to transfect of adult rodent retinal culture. In this method, eyes were enucleated, devoid of retinal pigment epithelium (RPE) and whole mount retina transfected with pGL3 and pGFP encoding plasmids.
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| Results:
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After 4 days of transfection luciferase activity was measured by luminometer. In less than 24 hours GFP expression was seen by fluorescence microscope in photoreceptor layer. Investigation with fluorescence microscope showed that not only the superior cells but also inner retinal layers transfected.
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| Conclusion:
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This method is the first report of retinal organotypic transfection with calcium phosphate in retinal layers which allow deep and better studies on function and regulation of gene expression in the retina. Feasibility, Safety, rapid, cost effective and reproducibility of this method makes it very valuable for retinal research.
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| Attachment:
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