(531i) Rapid and Multicycle Smfish Enabled By Electrokinetic Ion Concentration Polarization for Multiplexed in Situ Profiling of Tissue Specific Gene Expression in Whole C. Elegans

Regulation of gene expression plays a critical role in many physiological processes ranging from embryogenesis to development to aging. Studies of model organisms, such as Caenorhabditis elegans and Drosophila melanogaster, have shown that gene expression profile is highly heterogenous across different tissues and individuals. One promising technique to characterize gene expression with tissue and cellular specificity in whole organisms is single molecule fluorescent in situ hybridization (smFISH). Fluorescently labeled gene-specific probes are transported into the organism and hybridize to individual messenger RNAs (mRNAs), revealing the gene expression level and spatial localization. To analyze multiplexed gene expression patterns within the same animal, multiple rounds of probing, stripping, and re-probing are required while maintaining the individual identities of the animals. However, adapting multi-cycle smFISH by sequential probing is challenging in a whole organism due to inefficient probe exchange by diffusion-dominant transport across dense tissues.

To address this issue, we present a microfluidic/electrokinetic hybrid platform to enable rapid and multi-cycle smFISH in whole organisms, using C. elegans as a model. Our device integrates an array of individual animal traps gated by a cation-selective nanoporous membrane. Individual animals are isolated and held in place by microfluidic traps during the multiple rounds of smFISH probing, maintaining their individuality. Taking advantage of the charge selectivity of the cation-exchange membrane, we can build ion concentration polarization (ICP) by applying DC electric fields across the membrane and traps to achieve local isotachophoretic focusing of smFISH probes to enhance reagent exchange and probe hybridization. After each round of probing, we can completely remove the probes from in situ mRNAs, which is achieved through a locally focused electric field by establishing ion depletion zone in the animal traps and coupled with stripping agents. By rapidly transporting reagents across tissue barriers of the whole animal with field-enhanced electrophoresis, rather than diffusion, we are able to shorten the multiplexed smFISH process from days to a few hours.

We demonstrate the utility of this platform in an example studying dynamic gene expression with tissue-specific resolution. We profile an age-related, neuronally expressed gene, gpa-3, in adult C. elegans. Using the ICP-enhanced smFISH, we can reliably re-probe the expression of gpa-3 multiple times, demonstrating the robustness of our method. Along with probing the transcription of multiple genes within the same animal, we can incorporate other levels of information using our device; we demonstrate this by coupling the smFISH-based transcription profiling with fluorescent transcriptional reporters to quantify gene expression relationships on an individual level. Further we can directly compare protein and mRNA expression patterns of known age-related genes by adapting our platform to enhance immunofluorescence quantification of protein expressions in addition to the smFISH-based approach. This multi-level information enables us to study the heterogeneity in the decline of post-transcriptional regulation in aged animals. This method therefore lays the foundation to establish an individual-specific, system-wide, multi-level, and spatially resolvable gene expression map of whole organisms.