Optimizing Nanoporeters for Multiplexable Mammalian Reporting Systems | AIChE

Optimizing Nanoporeters for Multiplexable Mammalian Reporting Systems

Authors 

Nguyen, A. - Presenter, University of Washington
Wilde, D. - Presenter, University of Washington
Nivala, J., University of Washington
For over 40 years, reporter systems have been used as a means to track different types of cellular activities such as gene regulation. However, the number of uniquely addressable reporters that can be used together is limited due to their readout signal overlap. For instance, simultaneous measurement of unique genetic elements with multiple fluorescent protein variants is often obscured by overlapping spectral distributions. This prevents multiplexing, which not only impacts scalability and convenience, but also the potential complexity of the system of interest. To overcome this problem, we have previously designed a new class of reporter proteins, Nanopore-addressable protein Tags Engineered as Reporters (NanoporeTERs), that are more scalable and multiplexable than traditional reporter strategies and can be read out on a commercial nanopore array. Here, we optimized this system for mammalian cell systems and characterize 9 orthogonal barcodes which can be differentiated by analyzing their nanopore current signals with machine learning. We then demonstrate this system in HEK293 cells transfected with differentially barcoded genetic circuits. Ultimately, we aim to utilize this novel reporter system to investigate complex mammalian processes such as chromatin regulation dynamics and the determination of cellular phenotypes using customized reporter cassettes.