Construction And Phenotypic Investigation Of RIP1-D325A Mutant Mice

RIP1 is a key effector in TNF-induced necroptosis.The function of RIP1 is mediated by its serine/threonine-protein kinase activity and requires the collaboration of two downstream molecules,RIP3 and MLKL.Embryonic lethality caused by Caspase-8 deletion can be rescued by disrupting RIP1,RIP3 or MLKL,indicating that Caspase-8 can inhibit RIP1/RIP3/MLKL-dependent necroptosis to ensure successful embryogenesis.However,the molecular mechanism for this critical regulation remains unclear.One potential mechanism is that Caspase-8 inhibit necroptosis by cleaving RIP1.Our work aims to explore the physio-pathological role of RIP1 cleavage by Caspase-8.We generated a mutant mouse strain carrying RIP1 D325A mutation,which is the Caspase-8 cleavage site in RIP1,with the help of CRISPR-Cas9 system and haESCs technology.It was found that Rip1D325A/D325A mice die in midgestation,similar to Caspase-8-/-mice,but in sharp contrast to Rip1-/-mice which are normal during embryogenesis.Surprisingly,p-MLKL,the marker of necroptosis,can not be detected in Rip,D325A/D325A embryos,but cleaved Caspase-3,the marker of apoptosis,was observed in the early stage of yolk sac endothelial cells.Therefore,RipjD325/D325A embryos die of Caspase-3 mediated cell death in the yolk sac endothelial cells rather than RIP3/MLKL-dependent necroptosis.In summary,our data suggests that cleavage of RIP1 is critical during embryonic development and that RIPI D325A mutant is not simply a Caspase-8 non-cleavable mutant since Rip1D325A/D325A embryos can not phenocopy Caspase-8-/-mice.The death of Rip1D325A/D325A embryos is executed in a mechanism previously unappreciated.Future work will focus on exploring the detailed mechanisms in Rip1D325A/D325A embryonic lethality.