Journal of neuroscience methods · 2025
Development of high-content screening assay for gene silencing in adult sensory neurons.
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Canete Phillip, Do David, Lie Richard, Woodruff Grace, Gonzalez Rodrigo Lopez, Poplawski Gunnar
Аннотация
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.
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.