Bioactivities of culture supernatants from retroviral packaging cells carrying the mouse Fas ligand gene

Abstract

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.

Keywords

mFasL / retroviral packing cell / culture supernatant / bioactivity

References

[1] TakashisS, TomohiroT, PierreG, et al. . Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell, 1993, 751169-1169

[2] TomohiroT, MasatoT, CamilynnI, et al. . Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell, 1994, 76969-969

[3] MotomuS, AdrianoF, YasutakaT, et al. . Induction of antitumor immunity with Fas/Apo-1 ligand (CD95D-transfecfed neuroblastoma neuro-2a cells. J Immunol, 1999, 162(12): 7350-7350

[4] DonaldB, DanielG, HelenaS, et al. . A role for CD95 ligand in preventing graft rejection. Nature, 1995, 377630-630

[5] 2: 1999.631–649

[6] Sambroo K J, Fristsch E F, Maniatis T. 1995. 976–982

[7] AlbertoA, MichelB, ClaudeB, et al. . T cell recepor-induced Fas ligand expression in cytotoxic T lymphocyte clones is blocked by protein tyrosine kinase inhibitors and cyclsporin A. Eur Immunol, 1994, 242469-2469

[8] KotaniH, NewtonO B, ZhangS, et al. . Improved methods of retroviral vector transduction and production for gene-therapy. Hum Gene Ther, 1994, 519-19

[9] BurkhardH, JeanY C, PatriciaD S, et al. . High-efficiency retroviral transduction of mammalian cells on positively charged surfaces. Hum Gene Ther, 2000, 1143-43