Simultaneous, Large Multi-Gene Delivery for Implementation of Fluorescent Reporter Spatial Multiplexing to Image Signaling Pathways

Abstract

In order to study intricate, multifaceted biological systems, one must be able to measure numerous cellular activities in real-time. Today, many intracellular, genetically encoded reporters provide a readout in living cells using fluorescent proteins. Spectral multiplexing has been attempted by engineering reporters to each have a unique spectrum that does not overlap with other reporters to allow for simultaneous recording of multiple signals in a single physiological cascade. However, the problem with spectral multiplexing is the large degree of spectral overlap from the broad shape of the reporter’s spectrum that leads to a blended signal masking which data belongs to which molecule. This dissertation describes the concept of spatial multiplexing to circumvent the limitations of spectral multiplexing, the challenges of designing a system to deliver multiple reporters simultaneously in culture, and the attempt to teach resilience in developing tools for understanding the nervous system in a 14-week course.

By fusing a fluorescent reporter to a pair of self-assembling peptides or a self-assembling RNA-protein pair, reporters could be stably clustered within cells at random points, distant enough to be resolved by a microscope, but close enough to spatially sample the relevant biology. These clusters, called signaling reporter islands (SiRIs), can be modularly designed and permit a set of fluorescent reporters to be efficiently adapted for simultaneous measurement of multiple points in a signaling pathway within single cells. SiRIs for indicators of second messengers and kinases were created to image up to five signals at once in a single living neuron. This introduces the need to express multiple genes in the same cell, but the probability of multiple plasmids entering the same cell for simultaneous expression is lowered as the number of plasmids increases. Therefore, means of expressing multiple SiRI genes in a single plasmid backbone were evaluated. Lastly, a record is shared of the process used to develop and implement a digital learning experience for teaching the place of failure in research and development.