Bioactivities of culture supernatants from retroviral packaging cells carrying the mouse Fas ligand gene |
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Authors: | Liu Lingbo Zou Ping Guo Rong Xiao Juan Xu Zhiliang |
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Institution: | (1) Institute of Hematology, Xiehe Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430022 Wuhan |
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Abstract: | Summary The bioactivities of culture supernatants from retroviral packaging cells carrying the mouse Fas ligand (mFasL) gene was investigated.
FasLcDNA was cloned into PLXIN with an internal ribosome entry site to link two cistrons through gene recombination technology,
PLXIN and the recombinant vector PLFIN were separately transfected into PA317 retrovirus packing cell line by lipofectamine
2000, and the resistant clones were selected with G418 selective medium. The integration of genome DNA was assayed by genomic
DNA PCR. NIH3T3 cells were transduced by the culture supernatants from PA317 carrying the mFasLcDNA gene, and were selected
with G418 selective medium, so as to select the PLFIN-PA317 clone capable of producing higher titer of supernatants. The levels
of mFasL protein on N1H3T3 cells membrane were assayed by flow cytometry (FCM). The biological activity of mFasL on NIH3T3
cells membrane was investigated by the inducing apoptosis of Fas+ Yac-1 cells co-cultured with NIH3T3 cells expressing Fas ligand. To explore the direct mFasL cytotoxicity of culture supernatants
from retroviral packaging cells carrying the mFasL gene, the culture supernatants from PLFIN-PA317 and PLXIN-PA317 were separately
co-cultured with Yac-1 cells in parallel. The recombinant PLFIN was successfully constructed. The highest titer of supernatants
from twelve resistant clones was 8. 5 × 105 colony-forming-unit (CFU)/ml. The NIH3T3 cells transfected by above supernatants had a higher level of mFasL (53. 81 ±6.9
%), and significantly induced the apoptosis of Fas+ Yac-1 cells (56.78±4. 5 %), as both were cocultured for 5 h at 1:1 ratio, whereas it is 7. 08±3. 4 % in control group (P<0.01). Supernatant from PLFIN-PA317 could also directly induce the apoptosis of Yac-1 within 5 h of incubation. Thus, the
culture supernatants from PLFIN-PA317 possessed both infectivity and cytotoxicity of mFasL.
This project was supported by a grant from National Natural Sciences Foundation of China (No. 39770767). |
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Keywords: | mFasL retroviral packing cell culture supernatant bioactivity |
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