Zhao, Bojun and Allinson, Sarah L. and Ma, Aihua and Bentley, Adam J. and Martin, Francis L. and Fullwood, Nigel J. (2008) Targeted cornea limbal stem/progenitor cell transfection in an organ culture model. Investigative Ophthalmology & Visual Science, 49 (8). pp. 3395-3401. ISSN 0146-0404Full text not available from this repository.
PURPOSE. To optimize a nonviral gene transfection system targeting the corneal limbal stem/progenitor cells. METHODS. A plasmid containing LacZ gene coding for beta-galactosidase (beta-gal) was transfected into human corneal epithelial cells (HCECs) and multilineage progenitor cells (MLPCs) with different transfection reagents, to determine the optimal transfection reagent. In an ex vivo study, the bovine corneal epithelium and limbal stem/progenitor cells were transfected with a microinjection system with a 36-gauge needle that delivered plasmid/transfection reagent (Lipofectamine 2000; Invitrogen, Carlsbad, CA) complexes. The transfected corneoscleral discs were cultured in an air-interface culture system. The expression of (beta-gal) was determined with an X-gal staining assay, and images were acquired with light microscopy and transmission electron microscopy. The expression of cytokeratin K5/14 and K3/K12 in corneal and limbal epithelium was determined by immunohistochemistry. RESULTS. The highest percentages of beta-gal expression in HCECs and MLPCs were achieved when the transfection reagent Lipofectamine 2000 was used. Corneal epithelial and limbal basal cells were successfully transfected with the reporter gene by targeted microinjection of plasmid/liposomal complexes. The location of the bovine limbal stem/progenitor cells was confirmed by positive K5/K14 labeling and negative K3/12 labeling. CONCLUSIONS. Targeted microinjection of plasmid/liposomal complexes resulted in limbal stem/progenitor cell transfection. This technique has potential for the short-term treatment of corneal diseases.
|Journal or Publication Title:||Investigative Ophthalmology & Visual Science|
|Uncontrolled Keywords:||EPITHELIAL PROGENITOR CELLS ; STEM-CELLS ; GENE-THERAPY ; INFRARED MICROSPECTROSCOPY ; IN-VIVO ; EXPRESSION ; DELIVERY ; IDENTIFICATION ; RENEWAL ; VECTOR|
|Subjects:||Q Science > QH Natural history > QH301 Biology|
|Departments:||Faculty of Health and Medicine > Biomedical & Life Sciences|
|Deposited By:||Dr Sarah Allinson|
|Deposited On:||17 Jun 2008 16:46|
|Last Modified:||25 Feb 2014 10:06|
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