Creative Biolabs

Calcium Integrator FLiCRE Design and Construction

Calcium Integrator FLiCRE Design and Construction

In the brain, a network of functional neural cells coordinate their synaptic activity to process stimuli and cause specific behaviors. Recording transient cellular events is necessary to detect neurons activity and delineate the interconnected mechanism of behavioral output. However, the complexity of many functional cell networks makes it difficult to describe and characterize. There are few tools to answer basic questions, such as which interconnected cell types make up a given cell network and how the activity of these cells drives behavioral responses.

At Creative Biolabs, we have developed a "calcium molecular integrator", FLiCRE (Fast Light and Calcium-Regulated Expression), which can be used for optogenetic channel labeling of neural cells. It is known that the increase in intracellular calcium faithfully tracks action potentials in milliseconds rather than hours. Therefore, the monitoring of calcium-based signals provides a more time-specific and extensive method for activity-dependent cell labeling. The engineered tool FLiCRE enables the integration of the transient increase in intracellular calcium, and then delivers the calcium signal stably in the cell as a novel, modular transcript. FLiCRE holds sufficient specificity and resolution to achieve molecularly recording and and functional characterization of calcium channel signaling in the neural circuit.

Design of FLiCRE

Calcium is an almost-universal indicator of cell activity. FLiCRE consisists of two chains of components that respond to the existence of light signal and calcium in the cell. The two molecule components in the light sensitivity system are packaged in two parts by recombinant virus. To get into the cell, one of the part of FLiCRE expresses the protein (any gene is applicable) which can enter the cell. The other part of FLiCRE is designed for release the protein while the calcium concentration is changed and the cells are immersed in light. Once the protein is released and the gene is expressed, researchers can use RNA sequencing to quickly create cell-to-cell records that accurately record the neurons that FLiCRE has activated.

Schematic diagram of FLiCRE.Fig.1 Schematic diagram of FLiCRE. (Christina, 2020)

Advantages of FLiCRE

  • FLiCRE enables light and calcium gated molecular labeling of active neurons. The existing marking technology takes several hours to activate, while the FLiCRE marking process only takes a few minutes.
  • Circuit that combines FLiCRE and optogenetic tagging recognition function coupling
  • FLiCRE readout with single-cell RNA sequencing links cell type with activity
  • FLiCRE-driven opsin reveals the behavioral function of activated neurons
  • FLiCRE studies thousands of cells at a time, while other techniques often require the analysis of multiple microscopic images each containing hundreds of cells.

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Reference

  1. Kim, C. K., Sanchez, M. I., Hoerbelt, P., Fenno, L. E., Malenka, R. C., Deisseroth, K., & Ting, A. Y. (2020). A Molecular Calcium Integrator Reveals a Striatal Cell Type Driving Aversion. Cell.
For Research Use Only. Not For Clinical Use.
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