Name: localized rnai and ectopic gene expression in the medicinal leech
Uploaded: 2008-04-17T22:00:00
Description: Watch this Scientific Journal Video about Localized RNAi and Ectopic Gene Expression in the Medicinal Leech at JoVE.com

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.

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
<ol>
<li>Place 100% isopropanol on ice.</li>
<li>Resuspend 5 mg gold particles (S1600ri, Seashell Technology, LLC; average diameter 1.6 &micro;m) in 100 ml binding...

<ol>
	<li>Baker, M. W., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+of+the+receptor+tyrosine+phosphatase+HmLAR2+in+a+single+cell+of+an+intact+leech+embryo+leads+to+growth-cone+collapse.">RNAi of the receptor tyrosine phosphatase HmLAR2 in a single cell of an intact leech embryo leads to growth-cone collapse.</a> Current Biology. 10, 1071-1074 (2000).</li><li>Baker, M. W., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Hirudo+medicinalis+DNA+Promoters+for+Targeted+Gene+Expression.">Characterization of Hirudo medicinalis DNA Promoters for Targeted Gene Expression.</a> Journal of Neuroscience Methods. 156, 145-153 (2006).</li><li>Gan, W. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+expression+of+a+leech+netrin+suggests+roles+in+the+formation+of+longitudinal+nerve+tracts+and+in+regional+innervation+of+peripheral+targets.">Cellular expression of a leech netrin suggests roles in the formation of longitudinal nerve tracts and in regional innervation of peripheral targets.</a> Journal of Neurobiology. 40, 103-115 (1999).</li><li>Juven-Gershon, T., Cheng, S., Kadonaga, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+a+super+core+promoter+that+enhances+gene+expression.">Rational design of a super core promoter that enhances gene expression.</a> Nature Methods. 3, 917-922 (2006).</li><li>Rinberg, D., Simonnet, C., Groisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumatic+capillary+gun+for+ballistic+delivery+of+microparticles.">Pneumatic capillary gun for ballistic delivery of microparticles.</a> Applied Physics Letters. 87, 014103 (2005).</li><li>Shefi, O., Simonnet, C., Baker, M. W., Glass, J. R., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtargeted+gene+silencing+and+ectopic+expression+in+live+embryos+using+biolistic+delivery+with+a+pneumatic+capillary+gun.">Microtargeted gene silencing and ectopic expression in live embryos using biolistic delivery with a pneumatic capillary gun.</a> Journal of Neuroscience. 26, 6119-6123 (2006).</li></ol>

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 &#181;m and particles were found ~15 &#181;m around the mean penetration depth, which was adjustable between 0 and 50 &#181;m. 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...

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<td>gold particles</td>
<td>Other</td>
<td>Seashell Technology, LLC</td>
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<td>&nbsp;</td>
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<td>binding buffer</td>
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<td>Seashell Technology, LLC</td>
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localized rnai and ectopic gene expression in the medicinal leech

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 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. 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.

Watch this Scientific Journal Video about Localized RNAi and Ectopic Gene Expression in the Medicinal Leech at JoVE.com

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

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.

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 ...

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