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[DESCRIPTION]
<p>This protocol describes the knock-down of specific tRNA(s) in mammalian cells using short hairpin RNA (shRNA)-based RNA interference (RNAi). We have used this method successfully to silence specific tRNAs in in HeLa<sup>1</sup> and N2a cells<sup>2</sup>.</p>
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<p>References</p>
<p>1. Kirchner <em>et al</em>. Alteration of Protein Function by a Silent Polymorphism Linked to tRNA Abundance. <em>PLoS Biology. </em>2017, <em>in press.</em></p>
<p><em>2. </em>Girstmair <em>et al</em>. Depletion of Cognate Charged Transfer RNA Causes Translational Frameshifting within the Expanded CAG Stretch in Huntingtin. <em>Cell Reports</em>. 2013, 3(1):148-59. DOI: <a href="http://dx.doi.org/10.1016/j.celrep.2012.12.019" target="_blank">http://dx.doi.org/10.1016/j.celrep.2012.12.019</a></p>

[BEFORE_START]
<p>shRNA expression vectors can be generated using the pSUPER siRNA expression vector system from OligoEngine. The pSUPER plasmid contains unique BglII and HindIII restriction site which can be used for integration of anealed forward and reverse shRNA oligonucleotides. This positions the shRNA oligonucleotide immediately downstream of the polymerase-III H1-RNA gene promoter, enabling expression of the desired shRNA. A T5 (5 consecutive thymidines) termination signal follows immediately downstream of the HindIII cloning site. For more details please see the attached pSUPER manual from OligoEngine. </p>
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<p>The following example outlines the generation of a pSUPER-based shRNA expression vector targeting the human tRNA<sup>Thr</sup>(CGU):</p>
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<p>1.<em> shRNA design</em></p>
<p>Please see the attached document for exemplary targeting of the human tRNA<sup>Thr</sup>(CGU).</p>
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<p>2. <em>Annealing of shRNA DNA oligonucleotides</em></p>
<p>shRNA-forming oligonucleotides (see step 1.) can be ordered as single stranded DNA oligonucleotides which are then annealed to form a double-stranded DNA molecule. The shRNA encoding dsDNA oligonucleotide contains 5' overhangs corresponding to BglII and HindIII restriction sites.</p>
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<p>3. <em>Linearizing the pSUPER vector</em></p>
<p>Linearize the pSUPER vector using BglII and HindIII restriction enzymes and gel purify the cut vector.</p>
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<p>4. <em>Ligation and transformation</em></p>
<p>The shRNA-encoding dsDNA oligonucleotide is then cloned into the linearized pSUPER vector. Prior to transformation, the ligation mixture is treated with BglII to remove all incorrectly ligated products. Ligation mixtures are then transformed into competent <em>E. coli</em>.</p>
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<p><em>Note: The BglII 5' overhang of the shRNA encoding dsDNA oligonucleotide actually corresponds to a BamHI restriction site within the pSUPER vector, and thus destroys the BglII site upon ligation. Hence, correctly annealed constructs do not contain a BglII restriction site.</em></p>
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<p>5. <em>Selection of positive clones</em></p>
<p>Positive clones are indetified using EcoRI and HindIII restriction analysis or integration PCRs.</p>
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<p>6. <em>Purification of shRNA expression plasmids</em></p>
<p>Purify shRNA expression plasmids using the EndoFree Plasmid Maxi Kit (Qiagen, 12362) and resuspend the plasmid DNA in endotoxin-free H<sub>2</sub>O. Store purified plasmids at a concentration of 1 µg/µl at -20°C until use.</p>
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