Mixed lineage kinase domain-like pseudokinase (MLKL) is a protein than plays a vital role in necroptosis, a type of programmed cell death with morphological characteristics of necrosis. When phosphorylated, MLKL oligomerises, translocates to the cell membrane and causes cell death. A lot is already known about the process of oligomerisation; however, the structure of oligomer and the mechanism of its action remain to be determined. To learn more about MLKL and its mechanism, a fusion protein M33-mCherry, composed of M33 nanobody and red fluorescent protein mCherry in a pcDNA3.1 vector, had already been prepared. M33 is specific for the N terminal region of human MLKL, and together with mCherry forms a chromobody. In our study, we wanted to determine at what time, after transfection, the expression of a chromobody is the most prominent. First, we transfected HEK293 Flp-In cells that already have integrated MLKL, however, we did not induce its expression, as that would result in cell death. At certain time stamps after transfection, the cells were lysated and analysed, using SDS-PAGE and western blot. After 16 h and 48 h, the cells were also observed under a fluorescent microscope to determine the transfection efficiency and the localisation of chromobody inside the cell. Results showed that overall, the transfection efficiency was very low, despite some cells having high chromobody expression levels. In those cells, M33-mCherry mostly localised in a specific part of the cell. The results of western blot showed that the chromobody was partially degraded. We also concluded that the expression of chromobody did not change significantly in the first 24 h after transfection, however, there was an increase after 48 h. Future experiments could be optimised by exploiting the Flp‑In system, which allows for a stable integration of the desired DNA sequence at a specific genomic location. By stably integrating M33-mCherry, transfection could be avoided altogether, which would make future experiments easier to conduct.
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