THE ENHANCED GREEN FLUORESCENT PROTEIN AS A MARKER FOR HUMAN TUMOR CELLS LABELLED BY RETROVIRAL TRANSDUCTION

Efficient delivery of foreign genes into target cells is needed for gene marking and gene therapy. Since the inception of murine retroviral-mediated gene transfer, there has been an intense interest in gene marker systems that allow direct observation of transferred genes into living cells. Many selectable reporter molecules, such as the neomycin resistance gene NeoR, the bacterial b-galactosidase gene LacZ, the gene for human low-affinity nerve growth factor receptor (NGFR) as well as the …

The enhanced green fluorescent protein as a marker for human tumor cells labelled by retroviral transduction

Objective: To investigate the feasibility of marking the human tumor cells with enhanced green fluorescent protein (EGFP) in vitro. Methods: The retroviral vector LGSNencoding EGFP was constructed and three human tumor cell lines were infected with LGSN amphotropic virus.Tumor cell lines that stably express EGFP were selectedwith G418. The integration and expression of EGFP gene were analyzed by polymerase chain reaction, and flowcytometry (FCM). Results: After gene transfection andping-pong transduction, amphotropic producer lineAm12/LGSN was generated with a stable greenfluorescence signal readily detectable by FCM in up to 97% of examined cells. The viral titer in the supernatants was up to 8.2105CFU/ml. After transduction and selection,G418-resistant leukemia K562, mammary carcinomaMCF-7, and bladder cancer 5637 cells were developed, inwhich the integration of both EGFP and neomycinresistance gene was confirmed by DNA amplification. In comparison with uninfected cells, FCM analysis revealedEGFP expression in up to 90% (range 85.5%~90.0%) oftumor cells containing LGSN provirus. Conclusion: Theretroviral vector LGSN can effectively mark the humantumor cells with a stably EGFP expression which may be in studying tumor growth, metastasis and angiogenesis.