Microtargeted gene silencing and ectopic expression in live embryos using biolistic delivery with a pneumatic capillary gun.

Introduction Modulating gene expression, either by transfection or by RNA interference (RNAi), is a powerful means for studying the functions of essential genes during nervous system development and maintenance. Both approaches depend on the successful delivery of the corresponding nucleic acids into the targeted cells. Current methods for introducing DNA and RNA into cells and tissues include viral transformation, lipofection, electroporation, direct injection through microcapillaries, and biolistics. In the last technique, the molecules to be delivered are carried by micron-size particles of a heavy metal that are accelerated to high speeds by flow of a gas and launched into the target tissue (Klein et al., 1987). The substances injected into cells using the biolistics technique have included DNA (Klein et al., 1987; Mehier-Humbert and Guy, 2005), fluorescent dyes (Gan et al., 2000), and, most recently, double-stranded RNA (dsRNA) (Hon et al., 2004; Kim et al., 2005). Particle-mediated delivery is not sensitive to permeability of the cell membrane by specific reagents and lacks the potentially deleterious effects of viruses and lipofection. It is particularly advantageous for live tissue applications, because it does not rely on molecular diffusion within tissue and can target cells in internal layers. Nevertheless, the application area of the particle-mediated delivery has been limited by the current design of “gene guns” used for particle acceleration. These gene guns, including the table-top PDS-1000 and the popular hand-held Helios (both commercially available from Bio-Rad, Hercules, CA) that are powered by pulses of highly pressurized helium (He), deliver particles to large areas (square centimeters) with limited accuracy and reproducibility. In addition, the tissue targeted by a Helios gun may be damaged by the high-speed jet of He emerging from the gun nozzle. Recently, a new type of pneumatic gun has been introduced (Rinberg et al., 2005) (Fig. 1A), in which particles are injected into a continuous stream of high-speed He in a capillary tube and accelerated up to speeds of 300 – 400 m/s. The stream of He emerging from the capillary outlet is completely diverted into a coaxial capillary of larger diameter, to which vacuum is applied, while the particles continue in their trajectories and retain their high speeds. The area targeted by this pneumatic capillary gun (called “the gun” in the rest of the text) is defined by the size of a nozzle, a circle 150 m in diameter, at the end of the outer capillary. Here we describe the application of the capillary gun for fast, accurate, and highly localized biolistic delivery of dsRNA and plasmid DNA into muscle cells and central neurons in internal layers of live embryos of the leech, Hirudo medicinalis. The leech embryo is proving to be a useful model for studying the effects of changing expression levels of specific genes on morphology and function of the nervous system (Aisemberg et al., 1997; Baker and Macagno, 2000; Wang et al., 2005). We demonstrate silencing of expression of the axon guidance factor netrin by RNAi, as well as ectopic expression of actin tagged with a fluorescent protein, in small clusters of longitudinal muscle cells and central neurons. We also show the biolistic transfection and staining in monolayer cultures of mammalian cells. Finally, we carry out independent injection of two different lipophilic tracking dyes with a single capillary gun.