Development of high-content screening assay for gene silencing in adult sensory neurons

Abstract

Background

High-content screening in post-mitotic neurons faces challenges due to low transfection efficiency and expensive viral or electroporation methods. To accelerate discovery specifically in adult sensory neurons, we sought a scalable, low-cost platform that preserves neuronal health.

Method

We developed a 384-well lipid-based siRNA screening assay using adult EGFP-expressing Fischer 344 DRG neurons. Key optimizations included a systematic comparison of plate plastics and lipid/siRNA ratios and reagents, yielding maximal knock-down with minimal toxicity.

Results

Optimal conditions (0.12 μL reagent, 2.5 pmol siRNA/well) reduced EGFP fluorescence by ≥ 50 % in 45 % of neurons, with mean knockdown efficiencies up to 60 % and minimal impact on neurite length. PTEN-targeting siRNAs increased neurite outgrowth by 40 % (p < 0.001), while death siRNA reduced length by 30 % (p < 0.001), demonstrating sensitivity to both stimulatory and inhibitory gene perturbations.

Comparison

Our approach offers substantially lower cost and higher throughput than alternatives. Relative to electroporation protocols for adult DRG neurons, reagent cost is reduced ∼12-fold and hands-on time drops from ∼2 days to ∼3 h, while eliminating specialized equipment. Notably, the assay was optimized for cost-efficiency and scalability; for instance, our 384-well format protocol can screen hundreds of genes in triplicate for under $10,000, making high-content screening feasible in smaller laboratories.

Conclusions

This platform enables rapid, cost-effective evaluation of hundreds to thousands of candidate genes in adult sensory neurons, facilitating identification of neurite growth regulators.