Optimized Calcium-Phosphate-Based Co-transfection of Tau and tdTomato into Human iPSC-Derived Neurons for the Study of Intracellular Distribution of Wild-type and Mutant Human Tau.

Abstract

The study of Tau protein in disease-relevant neuronal cells in culture requires efficient delivery systems for transfection of exogenous Tau and also modulators and interactors of Tau. Transfection of cultivated cells using calcium phosphate precipitation is a simple and cost-effective approach, also for difficult-to-transfect and sensitive cells such as primary neurons. Because of its low cell toxicity and ease of use, the Ca2+-phosphate transfection method is one of the most widely used gene transfer procedures in neuroscience. However, Ca2+-phosphate transfection efficacy in neurons is poor, often in the range of 1-5%, limiting its use in functional investigations. Here, we outline our improved Ca2+-phosphate transfection methodology for human iPSC-derived neurons that yields a reasonable efficiency (20-30% for bright volume markers) without apparent effects on cell health. We have used it to introduce wild-type and mutant human Tau with and without co-transfection of a volume marker (used here: tdTomato). In sum, our procedure can deliver neuronal genes (e.g., MAPT) using typical eukaryotic expression vectors (e.g., using CMV promoter) and is optimized for transfection of human iPSC-derived neurons.