[Establishment of lentivirus-mediated system of double suicide genes and its killing effects on K562 cells].
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.
Jiang
Yi-Rong
YR
Department of Hematology, Qilu Hospital of Shandong University, Jinan 250012, China. jiangiirong1970@yahoo.com.cn
Liu
Chun-Sheng
CS
Chen
Xue-Liang
XL
Ma
Dao-Xin
DX
D043525:Cytosine Deaminase / Q000235:genetics*; D005437:Flucytosine / Q000494:pharmacology; D015774:Ganciclovir / Q000494:pharmacology; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D020014:K562 Cells; D013937:Thymidine Kinase / Q000235:genetics*
D005437:Flucytosine; D013937:Thymidine Kinase; D043525:Cytosine Deaminase; D015774:Ganciclovir