SCSB Lunch Series: Ian Wickersham, Ph.D.

Date: Friday, August 25, 2017
Time: 12:00pm – 1:00pm
Speaker: Ian Wickersham, Ph.D.
Affiliation: Principal Research Scientist, McGovern Institute for Brain Research, MIT

Talk Title: Cell-type-specific transgene expression in wild-type animals using endogenous proteins as skeletons for assembling split transcription factors
Abstract: Cell-type-specific manipulations, such as optogenetic or chemogenetic stimulation and inhibition, are critical for dissecting circuit function and dysfunction, but their use so far has almost entirely been restricted to genetically tractable species, in which genomic engineering – such as the insertion of the Cre gene into the parvalbumin locus – is used to restrict transgene expression to a functionally meaningful cell type of interest. Here we introduce a technology for causing expression of transgenes specifically within targeted cell types in wild-type animals. Engineered binding proteins specific to a targeted endogenous protein are separately fused to the two fragments of a split transcription factor. In the presence of the targeted protein, the fusion proteins bind to single molecules of the targeted protein, reconstituting a functional transcription factor complex which causes transgene expression from an exogenously introduced template.
Here we show that this approach can be used to achieve high expression of transgenes selectively in cells expressing calretinin, a calcium binding protein characteristic of important interneuronal subtypes in cerebral cortex, hippocampus, and other brain structures. Using the directed evolution technique of yeast surface display, we engineered binding proteins specific to calretinin, then constructed genetic fusions of the two binding proteins to various DNA binding domains and transcriptional activation domains. We show that the system is robust and modular, working with a variety of DNA binding domains and activation domains in various configurations. We show that the system can be delivered via adeno-associated viral vectors injected stereotaxically in order to cause targeted expression of EGFP or channelrhodopsin specifically in calretinin-expressing neurons in neocortex and hippocampus of wild-type mice and rats. Using whole-cell recordings in acute brain slices, we show that targeted fluorophore expression can be used to selectively record from the neuronal type of interest and that channelrhodopsin is expressed highly enough to allow optical control of the neurons’ activity.
This represents the first successful cell-type-specific transgene expression within any cortical interneuron subtype in wild-type animals of any species.