Showing papers on "Chemically induced dimerization published in 2012"

Rapid and orthogonal logic gating with a gibberellin-induced dimerization system

Abstract: Using a newly synthesized gibberellin analog containing an acetoxymethyl group (GA(3)-AM) and its binding proteins, we developed an efficient chemically inducible dimerization (CID) system that is completely orthogonal to existing rapamycin-mediated protein dimerization. Combining the two systems should allow applications that have been difficult or impossible with only one CID system. By using both chemical inputs (rapamycin and GA(3)-AM), we designed and synthesized Boolean logic gates in living mammalian cells. These gates produced output signals such as fluorescence and membrane ruffling on a timescale of seconds, substantially faster than earlier intracellular logic gates. The use of two orthogonal dimerization systems in the same cell also allows for finer modulation of protein perturbations than is possible with a single dimerizer.

Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells

Abstract: The main methods for producing genetically engineered cells use viral vectors for which safety issues and manufacturing costs remain a concern. In addition, selection of desired cells typically relies on the use of cytotoxic drugs with long culture times. Here, we introduce an efficient non-viral approach combining the Sleeping Beauty (SB) Transposon System with selective proliferation of engineered cells by chemically induced dimerization (CID) of growth factor receptors. Minicircles carrying a SB transposon cassette containing a reporter transgene and a gene for the F36VFGFR1 fusion protein were delivered to the hematopoietic cell line Ba/F3. Stably-transduced Ba/F3 cell populations with >98% purity were obtained within 1week using this positive selection strategy. Copy number analysis by quantitative PCR (qPCR) revealed that CID-selected cells contain on average higher copy numbers of transgenes than flow cytometry-selected cells, demonstrating selective advantage for cells with multiple transposon insertions. A diverse population of cells is present both before and after culture in CID media, although site-specific qPCR of transposon junctions show that population diversity is significantly reduced after selection due to preferential expansion of clones with multiple integration events. This non-viral, positive selection approach is an attractive alternative for producing engineered cells.

Acute manipulation of phosphoinositide levels in cells.

Abstract: Phosphoinositides are membrane-bound signaling phospholipids that function in a myriad of cellular processes, including membrane trafficking, cytoskeletal dynamics, ion channel and transporter function, and signal transduction. In order to better understand the role of phosphoinositides in cellular processes, different approaches to study the effects of the presence or absence of these lipids must be devised. Conventional approaches of manipulating phosphoinositide levels such as over-expression or genetic ablation of lipid enzymes cause prolonged exposure of the cells to changes in lipid levels that could result in compensatory actions by the cell or downstream alterations in cell physiology. In this chapter we present an approach used recently by various laboratories, including our own, to acutely manipulate phosphoinositide levels at target locations using chemically induced dimerization (CID) that can be spatially and temporally controlled. We discuss considerations when designing expression constructs for targeting specific cellular compartment membranes and present examples from the literature on different ways of perturbing phosphoinositide levels at particular organelle membranes using CID. In addition, we provide details on image acquisition, data collection, and data interpretation. CID technology can be applied to many lipid enzymes to broaden the understanding of the role lipid signaling plays in cell physiology.

Protein interface remodeling in a chemically induced protein dimer

Abstract: Although the development of chemically induced, self-assembled protein-based materials is rapidly expanding, methods for directing their assembly in solution are sparse, and problems of population heterogeneity remain. By exerting control over the assembly of advanced protein structures, new classes of ordered protein nanomaterials become feasible, affecting numerous applications ranging from therapeutics to nanostructural engineering. Focusing on a protein-based method for modulating the stability of a chemically induced dihydrofolate reductase (DHFR) dimer, we demonstrate the sensitivity of a methotrexate competition assay in determining the change in DHFR–DHFR binding cooperativity via interfacial mutations over a 1.3 kcal/mol range. This represents a change of more than 40% of the dimer complex binding energy conferred from protein–protein cooperativity (~3.1 kcal/mol). With the development of this investigative system and refinement of protein-based techniques for complex stability modulation, the directed assembly of protein nanomaterials into heterocomplexes and a concomitant decrease in population heterogeneity becomes a realizable goal. Copyright © 2012 John Wiley & Sons, Ltd.