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

Orit Shefi, Claire Simonnet, Michael W. Baker, James R. Glass, Eduardo R. Macagno, Alex Groisman

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

Abstract

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 μm in diameter, at the end of the outer capillary.
Original languageEnglish
Pages (from-to)6119-6123
Number of pages5
JournalJournal of Neuroscience
Volume26
Issue number23
DOIs
StatePublished - 7 Jun 2006
Externally publishedYes

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