Construction Of HT1080^RIP1-/- Cell Strain And MDA-MB-231^RIP1-/- Cell Strain By Using Transcription Activator-like Effector Nucleases

The most commonly used techniques to achieve gene knock out or knock down in cells includes RNA inference (RNAi),Zinc-Finger Nucleases(ZFN), Transcription Activator-like Effector Nucleases(TALEN) and the newly CRISPR-Cas 9(Clustered Regμlarly Interspaced Short Palindromic Repeats RNA). TALEN mediated gene knock out method get a very wide range of applications because of its high specificity, high targeting efficiency, low cost and convenient operation.Receptor-interacting protein 1 (RIP 1),which has the serine/threonine kinase activity, plays an important role in the regulation of apoptosis and cell apoptosis and necrosis. RIP1 includes the C-terminal kinase domain(KD) region, N-terminal death domain(DD) and intermediate domain(ID).ID area contains the RIP homotypic interaction motif(RHIM). RIP1 and RIP3 can combine through the interactions of RHIM to form a complex and then initiate cell apoptosis and necrosis. As an important adapter protein, RIP1 can assembled with a variety of cell surface receptors (such as TLR3, TLR4, TNFR1 and Fas, etc.)through the homotypic interaction motif to form cell death signaling complex. In addition,the cell apoptosis and necrosis were inhibited once the RIP1 kinase activity was inhibited, which means the importance of RIP1 kinase activity to cell apoptosis and necrosis, but the molecular mechanism is not clear. Therefore,it is very important to build RIP 1-deficient cell lines by TALEN,for the study of the molecular mechanisms of tumor cells drug resistance and the regulating mechanism of RIP1 in cell death.In this study, two cells lines,human fibrosarcoma cells HT1080 and human breast cancer cell MDA-MB-231, are utilised as the host cells and RIP1 as the target gene of TALEN. Firstly, it is important to assemble the TALEN targeting plasmid, and then after transfection,numbers of monoclonal screening work are carried out. In order to confirm the targeting cells, both of gene sequencing at the gene level and the western blot analysis at the protein expression level were employed. Through gene and protein expression levels tests, it is confirmed that RIP1 gene knock out monoclonal cell lines are finally achieved successfully, which can be used as RIP1 knoct out cells for the further study of RIP 1 function. The main results includes:1.The construction and activity examination of the RIP 1 TALEN targeting vector: two targeting sites are designed at the RIP1’s CDS region, and 10 TALEN arm plasmids are assembled through the classical module assembling method.After the preliminary digestion testing, these TALEN arm plasmids give a transfection to theHEK293T cells, the results show a high transfection efficiency up to 40%. In order to confirm the targeting activity of TALEN targeting plasmid,the transfected 293T cells were then sequenced, and the results show that the L1R1 arms work well and could successfully knock the RIP1 gene out.2.The RIP1 gene knockout of HT1080 by TALEN:HT1080 is a commonly used cell biology research experimental cell line,it is targeted by the L1R1 TALEN plasmid.Its transfection efficiency is about 15% by flow cytometry testing,after transfection,12 monoclonal cells were got through monoclonal screening.After genome sequencing,1 strain was successfully knocked out.In order to confirm the result, western blot were carried out. It confirms that RIP1 expression in stated cells is abolished. Thus HT1080RIP1-/-cells are successfully obtained.3. The RIP1 gene knockout of MDA-MB-231 by TALEN:In order to support the subsequent experimental studies sufficiently,it is necessary to build at least two or more RIP1-/-cell models. So once again,the MDA-MB-231 cells were targeted by L1R1 targeting plasmid.The transfection efficiency of the MDA-MB-231 cells was around 13%,and 10 monoclonal cells were got. And after the genome sequencing test and the western blot, it was confirmed both at the gene level and protein expression level that one strain of MDA-MB-231 RIP 1-/-cell got.The RIP1 gene was knockout at the cellular level by TALEN gene editing technology in this study. And successfully obtained two different cells of RIP 1-/-model,which are HT1080RIP1-/-and MDA-MB-231 RIP 1-/-.These are very good and suiteble cell models for the sudies of RIPTs phosphorylation status and function,which are also very valuble to clarify the role of RIP1 in the pathway of cell death and survivle, related to tumor pathology studies, as well as clinical research and the development of anticancer drugs.