Name: bacterial delivery of rnai effectors: transkingdom rnai
Uploaded: 2010-08-18T17:00:00
Description: Watch this Scientific Journal Video about Bacterial Delivery of RNAi Effectors: Transkingdom RNAi at JoVE.com

Work was supported by grant no. 01GU0615 of the "Bundesministerium f&uuml;r Forschung und Technologie (BMBF).

1) Bacterial Delivery of shRNAs
<ol>
<li> Before starting with the bacterial culture, one has to prepare LB-medium and LB-agar. </li>
<li> For the LB-medium weigh out yeast extract (0.5 % w/v), bacto tryptone (1.0 % w/v), and NaCl (0.6 % w/v) and dilute these components in aqua bidest. The prepared solutions have to be sterilized in an autoclave and are then ready to use. </li>
<li> For LB-agar plates bacto agar (1.5 % w/v) has to be added to the LB-medium previous to sterilization. </li>
<li> Heat up LB-agar in ...

<ol>
	<li>Kr&#252;hn, A., Wang, A., Fruehauf, J. H., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+short+hairpin+RNAs+by+transkingdom+RNA+interference+modulates+the+classical+ABCB1-mediated+multidrug-resistant+phenotype+of+cancer+cells.">Delivery of short hairpin RNAs by transkingdom RNA interference modulates the classical ABCB1-mediated multidrug-resistant phenotype of cancer cells.</a> Cell Cycle. 8, 3349-3354 (2009).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDR1/P-glycoprotein+(ABCB1)+as+target+for+RNA+interference-mediated+reversal+of+multidrug+resistance.">MDR1/P-glycoprotein (ABCB1) as target for RNA interference-mediated reversal of multidrug resistance.</a> Curr. Drug Targets. 7, 813-821 (2006).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+cancer+multidrug+resistance:+a+still+unsolved+problem.">An overview of cancer multidrug resistance: a still unsolved problem.</a> Cell. Mol. Life Sci. 65, 3145-3167 (2008).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+RNA+interference+in+drug-resistant+cancers.">Therapeutic potential of RNA interference in drug-resistant cancers.</a> Fut. Oncol. 5, 169-185 (2009).</li><li>Nguyen, T. A., Fruehauf, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transkingdom+RNA+interference+(tkRNAi):+a+novel+method+to+induce+therapeutic+gene+silencing.">Transkingdom RNA interference (tkRNAi): a novel method to induce therapeutic gene silencing.</a> Methods Mol. Biol. 514, 27-34 (2009).</li><li>Nieth, C., Priebsch, A., Stege, A., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+classical+multidrug+resistance+(MDR)+phenotype+by+RNA+interference+(RNAi.">Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi.</a> FEBS Lett. 545, 144-150 (2003).</li><li>Stege, A., Priebsch, A., Nieth, C., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+and+complete+overcoming+of+MDR1/P-glycoprotein-mediated+multidrug+resistance+in+human+gastric+carcinoma+cells+by+RNA+interference.">Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference.</a> Cancer Gene Ther. 11, 699-706 (2004).</li><li>Stein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+in+vivo+reversal+of+the+multidrug+resistance+(MDR)+phenotype+by+jet-injection+of+anti-MDR1+short+hairpin+RNA-encoding+plasmid+DNA.">Complete in vivo reversal of the multidrug resistance (MDR) phenotype by jet-injection of anti-MDR1 short hairpin RNA-encoding plasmid DNA.</a> Mol. Ther. 16, 178-186 (2008).</li><li>Xiang, S., Fruehauf, J., Li, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA-expressing+bacteria+elicit+RNA+interference+in+mammals.">Short hairpin RNA-expressing bacteria elicit RNA interference in mammals.</a> Nat. Biotechnol. 24, 697-702 (2006).</li></ol>

The cell number seeded for infection and the corresponding MOI used, critically depend on the cell lines under observation and their speed of growth. To find optimal cell numbers for seeding, pre-experiments to determine the speed of growth are strongly recommended. Besides this, different MOIs should be tested due to the limited extend to which the cells can stand the bacterial invasion without dying of stress. The optimal point of time where the down regulation of the gene under observation may vary. It is recommended ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>214050</td>
<td>&nbsp;</td>
<tr>
<td>Amphotericin B</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2612</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Phosphate Buffered Saline-PBS (10 x)</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>14080048</td>
<td>&nbsp;</td>
<tr>
<td>E. coli ceq221</td>
<td>&nbsp;</td>
<td>Cequent Pharmaceuticals Inc.</td>
<td>&nbsp;</td>
<td>No catalogue available, direct order</td>
</tr>
<tr>
<td>Gentamycin</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2710</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>&nbsp;</td>
<td>Contains 5,000 units of penicillin (base) and 5,000 &mu;g of streptomycin (base)/ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline.</td>
</tr>
<tr>
<td>Sodium chloride</td>
<td>&nbsp;</td>
<td>Merck KgaA</td>
<td>1064060500</td>
<td>&nbsp;</td>
<tr>
<td>Tryptone</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>211705</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>212750</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

bacterial delivery of rnai effectors: transkingdom rnai

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

Watch this Scientific Journal Video about Bacterial Delivery of RNAi Effectors: Transkingdom RNAi at JoVE.com

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

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