Liposome-based Expression of the PIEZO1 Sensor GenEPi in Hippocampal Neurons in Organotypic Slices.

Expressing large DNA constructs in the native three-dimensional brain microenvironment remains technically challenging. Although viral vectors provide high transduction efficiency and cell-type selectivity, their genetic payload capacity is limited. Various non-viral approaches have been used in brain tissue, but they may compromise tissue viability or require specialised equipment, such as biolistic delivery or electroporation. We present an adapted protocol for delivering the large DNA vector encoding the optical PIEZO1 sensor GenEPi into brain tissue to enable sensor expression in pyramidal neurons. By applying DNA-Lipofectamine liposomes directly to the slice surface, we achieved efficient, minimally invasive transfection of pyramidal neurons in the CA1 and CA3 regions of organotypic hippocampal slices. PIEZO1 sensor expression was detectable as early as 7 days after transfection, increased with longer tissue maintenance, and was sustained for 3-4 weeks in vitro. This protocol describes a cost-effective, non-invasive approach that preserves cell viability and enables investigation of PIEZO1-mediated mechanotransduction in a native brain microenvironment. Key features • DNA-Lipofectamine liposomes are applied directly to slice surface, enabling efficient transfection of superficial hippocampal neurons, important for imaging experiments performed using upright microscope systems. • The protocol provides a cost-effective gene delivery approach that requires only small volumes of DNA and transfection reagent. • Robust expression of the PIEZO1 sensor GenEPi is achieved within a relatively short time (approximately 1 week after transfection). • The method is compatible with long-term tissue maintenance, with neuronal viability and GenEPi expression maintained for up to 3-4 weeks after transfection.