Journal of the Korean Ophthalmological Society 2002;43(3):571-577.
Published online March 1, 2002.
Gene Transfer into Corneal Keratocytes using a Hybrid EBV/retroviral Vector.
Beom Jin Cho, Insuk Park, Mii Keum Kim, Won Ryang Wee, Jin Hak Lee
1Department of Ophthalmology, College of Medicine, University of Ulsan, Korea.
2Department of Ophthalmology, College of Medicine, Seoul National University, Korea. wrwee@snu.ac.kr
EBV/레트로바이러스 중합매개체를 이용한 각막간질세포에의 외부형질도입
조범진 ( Beom Jin Cho ) , 박인숙 ( In Suk Park ) , 김미금 ( Mii Keum Kim ) , 위원량 ( Won Ryang Wee ) , 이진학 ( Jin Hak Lee )
Abstract
PURPOSE
We tried to determine the feasibility and efficiency of foreign gene transfer into corneal keratocytes using a hybrid EBV/retroviral vector as an investigative trial for gene therapy in corneal diseases. METHODS: LZRSpBMN-Z, alac Z-transducing hybrid EBV/retroviral vector, was transfected into Phoenix(T M) amphotropic packaging cells based on a 293T cell line and then collected without/with puromycin selection (puro (-)/puro (+) vector respectively). Cultured human and rabbit keratocytes were transduced with lac-Z gene using the puro (-) or puro (+) vector solutions, then stained with 5-bromo-4-chloro-3-indolyl galactopyranoside (X-gal). FACS-Gal analysis of transduced corneal keratocytes was also performed for calculating gene transfer efficiency. In addition, as an in vivo trial, we tried to transduce rabbit keratocytes by topical application of the vector supernatants following PRK or lamellar dissection of rabbit corneas. RESULTS: In vitro, both cultred human and rabbit keratocytes were transduced successfully with lac - Z gene. Transduction efficiency was 22% and 16% for human and rabbit keratocytes respectively with puro (-) vector, and slightly increased to 24% and 22% with puro (+) vector. In vivo corneas, however, no keratocytes were stained with X-gal. CONCLUSIONS: A hybrid EBV/retroviral vector, LZRSpBMN-Z, successfully transduced corneal keratocytes in in vitro conditions but not in vivo corneas.
Key Words: Cornea;Keratocytes;Transduction;Gene therapy;EBV/retroviral vector


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