PubMed · 11262876
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
M Patterson. 2000. Immunogenetics. Not just another editor.. https://doi.org/10.1038/35049514
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
To establish lentivirus-mediated system of double suicide genes and explore its killing effects on K562 cells, lentivirus transfer vector for double suicide genes was constructed using molecular methods, three plasmids of lentivirus gene transfer vector system were transferred into packaging cell line 293T using lipofectine method, the transfer effect was observed through fluorescence microscopy, the lentivirus particles were observed by means of electron microscopy. High titer of lentivirus was harvested from the supernatant of virus-producing cell culture and concentrated by high-speed centrifugation with Poly-L-Lysine (PLL). The K562 cells were infected with the concentrated supernatant containing the virus with the double suicide genes. Fluorescence microscopy and RT- PCR confirmed the integration and expression of extraneous gene. The cytotoxicity to these transgenic cells treated with 5-FC and GCV was measured by MTT assays. The growth inhibition ratio (GIR) of cells and inhibition concentration 50 (IC(50)) were counted. After administration of GCV and 5-FC, the changes of those cells were observed through scanning electron microscope. The results showed that lentivirus transfer vector with double suicide genes was constructed successfully. The above-mentioned plasmids were effectively transferred into 293T cells. So much green fluorescence was observed through fluorescence microscope. A lot of lentivirus particles were observed through transmission electron microscope. Double suicide genes mediated by lentivirus were stably integrated and expressed in K562 cells after infection with the concentrated virus using fluorescence microscopy and RT-PCR. The GIR of K562 cells using GCV or 5-FC was 48.73% or 50.69% respectively and it was apparently higher than that of untransfected cells (P < 0.01). When using GCV and 5-FC together, the GIR was 87.69%, which was apparently higher than that of group using GCV or 5-FC alone (P < 0.01). In conclusion, lentivirus-mediated gene transfer system could transfer CD and TK double suicide genes into K562 cells with high efficiency and it had strong killing effects when giving 5-FC and/or GCV. The cytotoxic effects of double suicide genes were superior to that of single suicide gene. The lentivirus-mediated double suicide gene transfer system is a high-efficiency gene transfer vector.
BACKGROUND & OBJECTIVE: The lentiviral vectors can integrate interest genes into genome of the target cells that allow for stable transgenic expression even in non-dividing cells without evoking an immune response of the host. All the features have promised them to be used in vivo gene therapy. This study was designed to explore the killing effect of double suicide genes mediated by lentivirus on lymphoma cells (Raji). METHODS: The three plasmids expressed lentivirus, packaging plasmid pCMV 8.2, envelope plasmid pCMV.VSVG and target plasmid (pHR'CS. GFP as control group, pHR'CS.CDglytk as experiment group) were packaged into 293T cells using lipofectine method. Supernatant was harvested and concentrated. The Raji cells were infected with the concentrated virus. The gene integration and expression were confirmed by fluorescence microscopy and RT-PCR. After prodrug GCV or/and 5-FC administration, MTT method was used to detect the growth inhibition rate (GIR) of Raji cells for evaluating the killing effect of CD and HSV-tk double suicide genes on Raji cells. RESULTS: The three plasmids were effectively transferred into 293T cells. Green fluorescence on the cell was observed through fluorescence microscopy and a lot of virus particles were observed through transmission electronic microscopy. Double suicide genes mediated by lentivirus were effectively and stably expressed in Raji cells. The GIR of Raji cells using GCV or 5-FC was 51% or 50%, respectively, and it was apparently higher than that of untransfected cells(P< 0.01). When using GCV and 5-FC together, the GIR was 73%, which was apparently higher than that of group using GCV or 5-FC alone (P< 0.01). CONCLUSION: Double suicide genes mediated by lentiviral vector could transfect lymphoma cells effectively and stably. The double suicide gene system enhanced killing effect remarkably on lymphoma cells than CD/5FC or HSV-tk/GCV system alone.
The prodrug and antedrug concepts, which were developed to overcome the physical and pharmacological shortcomings of various therapeutic classes of agents, employ diametrically different metabolic transformations. The prodrug undergoes a predictable metabolic activation prior to exhibiting its pharmacological effects in a target tissue while the antedrug undergoes metabolic deactivation in the systemic circulation upon leaving a target tissue. An increased therapeutic index is the aspiration for both approaches in designing as well as evaluation criteria. The recent research endeavors of prodrugs include the gene-directed and antibody-directed enzymatic activation of a molecule in a targeted tissue, organ specific delivery, improved bioavailabilities of nucleosides and cellular penetration of nucleotides. As for antedrugs, emphasis in research has been based upon the design and synthesis of systemically inactive molecule by incorporating a metabolically labile functional group into an active molecule.