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Corneal epithelium as a platform for secretion of transgene products after transfection with liposomal gene eyedrops.
- Source :
-
The journal of gene medicine [J Gene Med] 2007 Mar; Vol. 9 (3), pp. 208-16. - Publication Year :
- 2007
-
Abstract
- Background: The first objective of the study was to evaluate the transfection of corneal epithelium with non-viral vectors to secrete transgene products into the tear fluid and aqueous humor. The second goal was to evaluate the differentiated corneal epithelial cell culture for transfection studies.<br />Methods: The human corneal epithelial (HCE) cell line was cultured to different stages of differentiation and transfected with complexes of pCMV-SEAP2 with DOTAP/DOPE, DOTAP/DOPE/protamine sulfate (PS) and polyethylenimine (PEI). The complexes of DOTAP/DOPE with plasmid (CMV-SEAP2 or pCMV-Luc4) were subsequently applied topically to the rabbit eyes. Secreted alkaline phosphatase (SEAP) was analyzed using chemiluminescent assay. Luciferase (Luc) was detected at the mRNA level in cornea and conjunctiva using a qRT-PCR.<br />Results: The transfection levels decreased with differentiation of HCE cells. PEI was effective in transfecting both the dividing and partly differentiated cells, but ineffective in differentiated cells. DOTAP/DOPE showed high activity in differentiated cell cultures, while added PS did not improve transfection. Significant SEAP expression was observed for three days after in vivo transfection in the tear fluid and aqueous humor. The luciferase mRNA was found both in the cornea and conjunctiva. The rates of SEAP secretion from both the basolateral side of differentiated HCE cells and cornea in vivo were within the same range.<br />Conclusions: Corneal epithelium can be transfected topically to secrete gene products to the tear fluid and aqueous humor. The differentiated HCE model is a useful tool in the evaluation of non-viral carriers for corneal transfection.<br /> (Copyright 2007 John Wiley & Sons, Ltd.)
- Subjects :
- Alkaline Phosphatase genetics
Alkaline Phosphatase metabolism
Animals
Cells, Cultured
Epithelium, Corneal chemistry
Eye metabolism
Fatty Acids, Monounsaturated chemistry
Humans
Luciferases genetics
Permeability
Phosphatidylethanolamines chemistry
Plasmids administration & dosage
Plasmids chemistry
Plasmids genetics
Quaternary Ammonium Compounds chemistry
RNA, Messenger analysis
RNA, Messenger metabolism
Rabbits
Tears metabolism
Epithelium, Corneal metabolism
Liposomes administration & dosage
Ophthalmic Solutions
Transfection methods
Transgenes
Subjects
Details
- Language :
- English
- ISSN :
- 1099-498X
- Volume :
- 9
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- The journal of gene medicine
- Publication Type :
- Academic Journal
- Accession number :
- 17351984
- Full Text :
- https://doi.org/10.1002/jgm.1011