In Vivo Injection of Synthetic Small Interfering RNA to Lethally Irradiated Mouse as a New Model to Study Apoptotic Pathways.

The Fas/Fas-ligand system is a well known component of the extrinsic apoptotic pathway. Using a short term culture assay we have established that CD34⁺ hematopoietic stem and progenitor cells express Fas antigen within 10 hours following irradiation. Using the terminaldeoxynucleotidyl transferase test, we have also established that this expression was linked with apoptosis since only the Fas/Fas-ligand positive cells exhibited a high level of DNA fragmentation (Drouet et al, Stem cell 1999). However Fas is also involved in the CD34⁺ cells differentiation process as described in ex vivo expansion studies. Caspases are other important actors of radiation induced (RI) apoptosis process and our team has recently identified caspases one and six as key actors in RI apoptosis in CD34⁺ cells. The goal of the present study was to evaluate short term synthetic small interfering RNA (siRNA) as new tools to in vivo modulate apoptosis in order to allow pathophysiological studies at the hematopoietic niche level. Briefly, B6D2F1 mice were globally irradiated (9 Gy gamma, LD90% 30 days) and then injected at 2 hours following irradiation with siRNA (0.5 nmol/mice, Dharmacon). To ensure a proper delivery to the niche cell components, siRNA were intra-tibially injected under a volume of 30μl. The duration of gene inhibition is about 10 days long. Control mice were injected with non relevant mock siRNA (n=80). Treated animals were injected with Fas-siRNA (n=20) or Fas + a pool of siRNA against caspases 1+ 3 + 6 + 8)(n=20). All mice were given ciprofloxacin during a week and no early lethality was observed. The lethality curves show that animals treated with Fas-siRNA exhibited an accelerated death rate when compared with siRNA mice. These results are compatible with a janus model for Fas expression depending on the time following irradiation: initial proapoptotic role, then requirement for cell expansion. Globally this study suggests the feasibility of using synthetic siRNA in vivo to screen the role of apoptosis actors.