An Introduction to Experiments Designing

The design of iPSC Safeguard pathway is simple and elegant. Basically, we can divide them into three major devices—the Suicide Gene, the MicroRNA-Target system and the Tet-off system, which has been described in detail in the Design module. For each device we had several candidates and before we finally assemble them into the whole pathway, we decided to test and characterize them carefully in the first place. The accurately quantitated parameters of each device then helped us set up a model and predicted which assembly scheme would work out best.

In order to test the devices, elements were cloned into our two major plasmids backbone: pcDNA3.0 and p199.The maps or design are shown in each parts of Results. We drove the Suicide Gene with CMV promoter to ensure a robust expression and prominent phenotype in cells. An eGFP was used to indicate the performance of Tet-off and MicroRNA-Target system, which could be quantitated through Image J and Western-Blot. All separately testing experiments were carried out via transient transfection in Bosc and HepG2, considering both the transfection efficiency and representation of liver cancer cell. Also, a survival experiment was done on liver cell to ensure that its miR122 level can successfully knockdown Suicide Gene with miR122 target-site.

After confirmation of each device, we proceeded to working on iPSC and assembly of the whole pathway. The whole pathway was integrated into two p199 plasmids, one for regulating and one for response .Lenti-virus packaging these two plasmids was produced and 3 cell lines of iPSC, HepG2, Hela were transfected. This successfully gave us the stable cell line with our iPSC Safeguard design. Then further test and characterization of the pathway’s performance of working as a whole were carried out. All the data can be seen in >>>>>.