Project description:A dominant-negative gene therapy approach has been proposed and tested on proto-oncogene KRAS, wherein the oncogenic activity (and cell proliferation) of KRAS can be suppressed by introducing a dominant-negative KRAS allele (S17N). We employed REPLACE to conduct continuous evolution on KRAS (S17N) and examined its potential pathways for conferring resistance in this gene therapy methodology.
Project description:A dominant-negative gene therapy approach has been proposed and tested on proto-oncogene KRAS, wherein the oncogenic activity (and cell proliferation) of KRAS can be suppressed by introducing a dominant-negative KRAS allele (S17N). We employed REPLACE to conduct continuous evolution on KRAS (S17N) and examined its potential pathways for conferring resistance in this gene therapy methodology.To explore the accumulation of mutations in various RNAs during the KRAS (S17N) evolution experiment, we established a barcoded library and conducted lineage tracing of replicative RNAs carrying KRAS (S17N) throughout the evolution process.
Project description:Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. Using REPLACE, we attempted continuous intracellular evolution of the negative dominant mutant KRAS (S17N). To analyze the process of mutation accumulation, we performed amplicon sequencing on experimental materials at different stages and under different treatment conditions. The results indicated that the mutations generated by this system were primarily induced by Monanunavir, and the addition of Monanunavir significantly accelerated the rate of evolution.
Project description:Transcriptional regulators are fundamental elements in synthetic biology. We sought to use REPLACE to evolve synthetic transcriptional regulators. Through weeks of directed evolution experiments, we succesufuly modified the sensitivity of TetR and PadR to ligand molecules.
Project description:Dominant negative mutatons in nuclear receptors can repress target gene expression, thus having stronger effects than simple loss of gene function. We sought to characterise the effects of a dominant negative mutant (p.W443R) in the hepatoma cell line HepG2 using cell lines stably transfected with wild type and mutant LXR isoforms.
Project description:Expression of a dominant negative HSF1 mutant (HSF379) was induced in HEK293 cells and the effect of the transcriptome of non-stressed cells was monitored
Project description:To assess cellular changes upon abrogation of the WNT-signaling pathway, we induced expression of a dominant-negative T-cell factor 4 (TCF4). This showed a remarkable overlap with activation of the unfolded protein response (UTR) in the same colon cancer cell line.