TY - JOUR
T1 - Retroviral vector-mediated gene transfer into keratocytes
T2 - In vitro effects of polybrene and protamine sulfate
AU - Seitz, Berthold
AU - Baktanian, Edwin
AU - Gordon, Erlinda M.
AU - Anderson, W. French
AU - LaBree, Laurie
AU - McDonnell, Peter J.
N1 - Funding Information:
Acknowledgements The b-galactosidase expression vector was kindly provided as high-titer PA317 packaging cell clones by Genetic Therapy, Inc., Gaithersburg, Md. The work reported here was supported in part by an unrestricted grant from Research to Prevent Blindness Inc., New York, N.Y., to the Department of Ophthalmology; by NIH Core Grant EY03040, Bethesda, Md.; and by Deutsche Forschungsgemeinschaft DFG-Az Se 667/5-1, Bonn, Germany. Dr. McDonnell is a Research to Prevent Blindness William and Mary Greve International Research Scholar.
PY - 1998/8
Y1 - 1998/8
N2 - Background: To determine the potential of somatic gene transfer as a novel technique for modulating corneal wound healing on a cellular level, the successful transduction of human keratocytes should be ascertained in vitro. In addition, the ability of different polycations to increase the transduction efficiency and their antiproliferative and cytotoxic effects should be assessed. Methods: To test transduction efficiency (X-Gal staining), cultured human keratocytes were incubated for 2 h with a retroviral vector bearing the β-galactosidase gene, with and without the addition of polybrene or protamine sulfate. To test the antiproliferative and cytotoxic effects, cultured human keratocytes were incubated with various concentrations of polybrene and protamine sulfate (0.08 to 800 μg/ml) for 2, 24 and 72 h, and evaluations were performed by means of an XTT-based colorimetric assay and phase-contrast microscopy. Results: Human keratocytes in vitro were transduced successfully with the β-galactosidase gene (3.5 ± 1.0%). Transduction efficiency was significantly (P ≤ 0.01) improved by addition of a polycation (from 12.3 ± 1.7% to 18.6 ± 2.3%), but there was no significant difference between the effects of polybrene and these of protamine sulfate. Both drugs induced a highly significant dose-dependent inhibition of proliferation (P < 0.001). ID50 ranged from 11 to 22 μg/ml with polybrene and from 15 to 244 μg/ml with protamine sulfate. Only with doses of 80 and 800 μg/ml did protamine sulfate produce less antiproliferative effects than polybrene (P ≤ 0.04). The lowest concentrations induced no morphological signs of cytotoxicity, whereas these signs were mild at 8 μg/ml and moderate to severe at the highest concentrations. Conclusions: Both polybrene and protamine sulfate can significantly improve the in vitro efficiency of successful retroviral vector-mediated gene transfer into keratocytes. Mild cytotoxic and moderate antiproliferative effects are to be expected in cultured keratocytes with a standard transduction procedure (8 μg/ml for 2 h).
AB - Background: To determine the potential of somatic gene transfer as a novel technique for modulating corneal wound healing on a cellular level, the successful transduction of human keratocytes should be ascertained in vitro. In addition, the ability of different polycations to increase the transduction efficiency and their antiproliferative and cytotoxic effects should be assessed. Methods: To test transduction efficiency (X-Gal staining), cultured human keratocytes were incubated for 2 h with a retroviral vector bearing the β-galactosidase gene, with and without the addition of polybrene or protamine sulfate. To test the antiproliferative and cytotoxic effects, cultured human keratocytes were incubated with various concentrations of polybrene and protamine sulfate (0.08 to 800 μg/ml) for 2, 24 and 72 h, and evaluations were performed by means of an XTT-based colorimetric assay and phase-contrast microscopy. Results: Human keratocytes in vitro were transduced successfully with the β-galactosidase gene (3.5 ± 1.0%). Transduction efficiency was significantly (P ≤ 0.01) improved by addition of a polycation (from 12.3 ± 1.7% to 18.6 ± 2.3%), but there was no significant difference between the effects of polybrene and these of protamine sulfate. Both drugs induced a highly significant dose-dependent inhibition of proliferation (P < 0.001). ID50 ranged from 11 to 22 μg/ml with polybrene and from 15 to 244 μg/ml with protamine sulfate. Only with doses of 80 and 800 μg/ml did protamine sulfate produce less antiproliferative effects than polybrene (P ≤ 0.04). The lowest concentrations induced no morphological signs of cytotoxicity, whereas these signs were mild at 8 μg/ml and moderate to severe at the highest concentrations. Conclusions: Both polybrene and protamine sulfate can significantly improve the in vitro efficiency of successful retroviral vector-mediated gene transfer into keratocytes. Mild cytotoxic and moderate antiproliferative effects are to be expected in cultured keratocytes with a standard transduction procedure (8 μg/ml for 2 h).
UR - http://www.scopus.com/inward/record.url?scp=0031822025&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0031822025&partnerID=8YFLogxK
U2 - 10.1007/s004170050129
DO - 10.1007/s004170050129
M3 - Article
C2 - 9717657
AN - SCOPUS:0031822025
SN - 0721-832X
VL - 236
SP - 602
EP - 612
JO - Graefe's Archive for Clinical and Experimental Ophthalmology
JF - Graefe's Archive for Clinical and Experimental Ophthalmology
IS - 8
ER -