Construction Of Cell Line For Screening Anti-cancer Drugs Targeted On Endogenous C-FLIP By CRISP-Cas9

Only the proliferation,division and apoptosis of cells can coordinate with each other in order to maintain the growth balance of normal tissues.If apoptosis is inhibited,the balance will be broken and the cell death rate will be reduced;If the body can not be restored to regulate,it will show an unlimited proliferation,and ultimately the formation of tumor.However,malignant tumors are characterized by high mortality in the world,the method is effective in the treatment of cancer and cure is a problem to be solved urgently.TNF related apoptosis inducing ligand is a member of the tumor necrosis factor superfamily newly discovered,It can selectively induces apoptosis of tumor cells without apparent toxic effects on normal cells.TRAIL has been a focus of intensive research.TRAIL is an ideal candidate target for the treatment of cancer.But TRAIL is a biological protein,consequently the high production costs,incovenient to save and transport,difficult to administer ece,so we can find the analogs of TRAIL is very necessary.Yunnan is rich in natural medicine resource.If some substance similar toTRAIL 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 patients.But the techique barrer is high to identify and obtain the active compounds from so many complex components in nature poducts.According to the ligand-receptor-effector fluorescence imaging(REFI)technique invented by Canon for our difficulties provides a new thinking.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 TRAILmimics.TRAIL induced apoptosis pathway has been well established.The TRAIL binds to death receotors4/5(DR4/5)and activated receptors recruit Fas-Associated protein FADD and procaspase8 through the interaction between death domains(DD)of DR4/5 and FADD,and between death efective domains(DED)of FADD and cFLIP,thereby inducing the formation of a large complex.We successfully constructed PCMV6-cFLIPsK192,195L,△ 203-221.GFP vector using molecular biological techniques,then it was transfected into U20S cells.Stably expressing PCMV6-cFLIPsK192,195L,:△203-221.GFP fusion protein in cell line was gained.The cells were treated with TRAIL,found that green green fluorescencae results was the same in the negative control and experimental group.Therefore,this cell line can not be used for the screening of natural drugs.We found that c-FLIPs A56 play a role in making c-FLIP combination with IKK.So it was mutated to leucine.Then the vectors was transfected to HEK293 cells,obtains two kinds of cell lines,fluorescence enter the nuclei and does not.Afer treated with TRAIL,Finally,found that fluorescence of both cells did not gathered.cFLIPs was reported to be recruited to FADD by its DED2 domain.DED family proteins exsit extensively in many cell lines,such as Procaspase-8,cFLIPL,cFLIPs etc,so that the exogenous c-FLIPs-GFP can not be recruited.So based on the endogenous cFLIP may be a better choice to construct this cell line.Using the third generation of gene editing technology-CRISPR-Cas9,GFP protein will be inserted in the exton5 or exton8 of cFLIP,so that cells express cFLIPs-GFP fusion protein.Experimental contents and results:1.Based on the exon 5 of cFLIPs,we constructed Guide-RNA and Donor DNA vector,then they were transfected into HEK293 cells by Electroporation Technology.We found that 10%of the cells can stable expression of cFLIPs-GFP fusion protein,but in the training process found that the expression of green fluorescent protein cells did not proliferate.It has been reported that K192 and 195L of the c-FLIPs will be ubiquitination,and its specific C tail structure can promote the degradation of c-FLIPs.While c-FLIPL does not occur this phenomenon,and then select the eighth exon of c-FLIPL gene was used as a target GFP of will be inserted.2.Based on the exon 8 of cFLIP,we constructed Guide-RNA vector and c-FLIP(8)-GFP fragments,then they were transfected into HEK293 cells.We screened a monoclonal cell line wich can stable expression cFLIPs-GFP fusion protein.The monoclonal cell lines were deal with TRAIL and we found no green fluorescence condensation in the cell line.3.The cells contain a large number of similar c-FLIPs DED family proteins such as Procaspase-8,so based on the currently constructed monoclonal cells may be a better choice to knock down the endogenous Procaspase-8.The currently constructed monoclonal cell lines cannot be used for follow-up screening,and we need further optimization it.