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1Division of Biological Sciences, University of California San Diego - UCSD, 2Department of Physics, University of California San Diego - UCSD
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In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.
Shefi, O., Simonnet, C., Groisman, A., Macagno, E. R. Localized RNAi and Ectopic Gene Expression in the Medicinal Leech. J. Vis. Exp. (14), e697, doi:10.3791/697 (2008).
In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 µm and extends over ~15 µm around the mean penetration depth, which is adjustable between 0 and 50 µm. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.
Particles can be coated with different reagents, such as dsRNA molecules, DNA plasmids or dyes. The type and diameter of particles are chosen according to the reagent and the depth of the target cells in the tissue: larger particles penetrate further, but may cause some damage to the tissue.
1. Preparation of dsRNA coated particles
2. Preparing the experimental setup for a specific assay
3. Loading the gun with the pre-coated particles
Particles should be loaded as a dry powder into the tygon tubing particle injection lines connected to the manifold. This tubing, which should be kept in a dry atmosphere at 4°C when not in use, can be re-used in additional experiments with the same reagents. Our experimental setup can deliver up to two different reagents per assay through separate ports in the manifold.
4. Animal Preparation
Before and after the gold particle delivery, the embryos are kept in sterile artificial pond water at room temperature.
5. Delivery of coated particles into the target tissue
One load of particles can typically be used for up to ten shots, with a single shot usually delivering on the order of a few hundred particles.
6. Immunostaining and neuronal labeling
Keep the embryos at room temperature and in darkness for 1 to 3 days after the particle delivery, until RNAi (or ectopic expression) is achieved. We then carry out one of the following procedures on the experimental specimens and controls:
In this demonstration, we used the pneumatic capillary gun to deliver gold particles into cells in small localized volume of a live leech embryo to knock-in and knock-down genes. The target region generally had a diameter of ~150 µm and particles were found ~15 µm around the mean penetration depth, which was adjustable between 0 and 50 µm. When the particles were coated with dsRNA molecules of the axon guidance factor netrin, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles. Particles coated with a plasmid encoding GFP induced fluorescence in neuronal cells when they stopped in their nuclei.
We have shown you how the pneumatic capillary gun gives the technique of biolistic delivery a new capability, to target microscopic regions of a tissue confined in three dimensions and targeted with high precision, without detectable damage to the tissue.
We would like to thank Dr. Michael Baker for his help in generating constructs for gene knock-in and for providing the time-lapse movies of growing sensory projections. We would also like to thank Virginia Vandelinder for technical assistance with the pneumatic gun experiments.
|gold particles||Other||Seashell Technology, LLC||S1600ri|
|binding buffer||Reagent||Seashell Technology, LLC|
|silicone rubber||Reagent||Dow Corning||Sylgard 184|
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