Original Article
HIGH EFFICIENCY RETROVIRUS-MEDIATED GENE TRANSFER TO LEUKEMIA CELLS
Abstract
Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia.
Method: The retroviral vector LXSN, containing the neomycin resistance (Neo R) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1. The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells.
Results: The titer of DOSPER directly transfected GP+envAml2 cells determined on NIH3T3 cells was 8.0x10 s CFU/ml, while that of producer infected with retrovirus was 1.6×107 CFU/ml. Integration of Neo R gene into all leukemia cells was confirmed by polymerase chain reaction (PCR). Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated Neo R gene on colonies after 7 days' culture.
Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.
Method: The retroviral vector LXSN, containing the neomycin resistance (Neo R) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1. The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells.
Results: The titer of DOSPER directly transfected GP+envAml2 cells determined on NIH3T3 cells was 8.0x10 s CFU/ml, while that of producer infected with retrovirus was 1.6×107 CFU/ml. Integration of Neo R gene into all leukemia cells was confirmed by polymerase chain reaction (PCR). Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated Neo R gene on colonies after 7 days' culture.
Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.
