Purpose To study the possibility of prevention of proliferative vitreoretinopathy（PVR） by transduction of exogenous gene in vivo． Methods PVR model of rabbits was induced by intravitreal injection of fibroblasts．beta;-galactosidase (lacZ) gene as a reporter gene was transfered into the vitreous of PVR model eyes mediated by retroviral vector, and the expression of the gene in eye tissues was determined ． Gene transfection was done on the 6th day after fibroblasts injection,and the dosage of intravitreal injection of reporter gene was 0．1ml PLXSN/lacZ serum-free supernatant (1．1times;106 cfu/ml)． Results lacZ gene expression was seen in proliferative membranes after gene transfection, and the expression was located maily at the surface of PVR membrane．The reporter gene expression lasted at least more than 30 days．No expression was found in retinal tissues． Conclusions Retrovirus mediated gene can be directionally transducted in PVR membrane,and might possess the feasibility of gene therapy for PVR． (Chin J Ocul Fundus Dis, 2001,17:224-226)
ObjectiveTo construct a luciferase reporter fusion containing the human connective tissue growth factor (CTGF) gene promoter.MethodsThe promoter region of the human CTGF gene (-835/+214) was amplified by polymerase chain reaction (PCR) using specially-designed primers, and subsequently cloned into the pGL3.0-Basic vector. Following screening and verification by single colony PCR, double digestion, and sequencing, the resulting pGL3.0-Basic-CTGF was used to transfect the human embryonic kidney cells 293T, human bronchial epithelial cells HBE and human lung epithelial cells A549, and its function in each cell line was determined by luciferase assay.ResultsSequence alignment showed 99.5% identity, suggesting successful construction of the pGL3.0-Basic-CTGF reporter fusion. Promoter activities were detected 48 hours after transfection of pGL3.0-Basic-CTGF into the 293T, HBE, and A549 cells, and the promoter activities were 2.416, 0.027, and 0.121, respectively (P<0.01). Moreover, the luciferase activity in the A549 cells was statistically higher than that in the HBE cells (P<0.01).ConclusionsThe human pGL3.0-Basic-CTGF luciferase reporter fusion has been successfully constructed. The construct exhibits promoter activity in the bronchial epithelial cells HBE and the lung epithelial cells A549, and can therefore serve as a useful tool for future research in transcriptional regulation.