Construction Of A Tumor-specific Drug Screening Cell Line Characterized By The Anti-apoptotic Protein C-FLIP_

Yunnan is rich in natural medicinal resources.If some substance similar to TRAIL is discovered and used to replace TRAIL,it will excel TRAIL and make the cost far lower,so as to alleviate the burden of cancer sufferers.But the technique barrier is high to identify and obtain the active compounds from so many complex components in natural products.According to the ligand-receptor-effector fluorescence imaging(REFI)technique,cell lines contain the fluorescence protein(GFP)fusion protein obtained by constructing a stable expression of this fusion protein can be used to screen compounds of TRAIL mimics.TRAIL induced apoptosis pathway has been well established.TRAIL binds to its death receptors 4/5(DR5/4)and the activated receptors recruits Fas-Associated protein FADD and c-FLIP through the interaction between death domains(DD)of FADD and c-FLIP,thereby inducing the formation of a large complex.By stably expressing a c-FLIPs-GFP fusion protein in cells,in theory,TRAIL treatment induces a change in fluorescence distribution in cell that can be visualized under fluorescence microscopy before and after TRAIL treatment.Aggregations or clusters of fluorescence occur in cell surface upon the formation of a large complex.The redistribution of fluorescence in cells can be analyzed and quantified.This technique can be used to screen TRAIL mimics in natural products.Experimental contents and results:1.Constructed pCMV6-c-FLIPSK192,195L,Δ203-221-GFP vector using molecular biological techniques,then it was transfected into U2OS cells.Stable expression of cFLIPSK192,195L,Δ203-221-GFP fusion protein in cell line was gained.The cells were treated with TRAIL,found that green fluorescence have gathered,but the result was the same in the negative control and experimental group.Thus constructed cell line is not our expectation.After the analysis of experimental results and the literature review found c-FLIPs A56 play a role in making c-FLIPs combination with IKK γ.2.c-FLIPs was reported to be recruited to FADD by its DED2 domain.Therefore,GFP fused to the c-terminal of DED2 may affect recruitment of c-FLIPs.In addition,A56 was reported to be responsible for the association between c-FLIPs and IKKγ,so it was mutated to leucine.Then the vector was transfected to HEK293 cells,obtained two kinds of stable cell lines,fluorescence enter the nuclei and does not.After treated with TRAIL,found that fluorescence of both cells didn’t gathered.3.DED family proteins exsit extensively in many cell lines,such as procaspase-8,c-FLIPL,c-FLIPs etc,so that the exogenous c-FLIPS-GFP protein can not be recruited.So based on the endogenous c-FLIPS may be a better choice to construct this cell line.Using the third genome-editing technique—CRISPR-Cas9,GFP protein will be inserted in the exon 5 of c-FLIPS.The related vectors were constructed recently,cell experiments are begining soon.