This work was supported by grants from Scuola Normale Superiore (SNS14_B_DIPRIMIO; SNS16_B_DIPRIMIO).

1. Cell Culture
<ol>
	<li>Culture SH-SY5Y cells (human neuroblastoma cell line, CRL-2266) in complete medium (Dulbecco&#39;s modified Eagle medium:nutrient mixture F12 [DMEM/F-12] supplemented with 10% fetal bovine serum [FBS], 2 mM L-glutamine, 100 U/mL penicillin and 100 &#181;g/mL streptomycin). Maintain the cells in an incubator at 37 &#176;C and 5% CO2. Grow cells in 10 cm plates and split when confluent.</li>
</ol>
2. Cell Differentiation
<ol>
	<li>To differentiate SH-SY5Y cells, the day after the plating, add 10 &#181;M retinoic acid (RA) to complete medium for 5 days.</li>
	<li>The sixth day replace medium with differentiation medium: DMEM/F-12 supplemented with 50 ng/mL BDNF, 2 mM L-glutamine. Do not add FBS or antibiotics.</li>
	<li>Grow the cells in differentiation medium for 3 days.</li>
</ol>
3. Chimeric Constructs Cloning
<ol>
	<li>Generate Tau-NLS construct by cloning by restriction enzyme digestion in frame at the 3&#39; end of Tau sequence 0N4R (383aa) the 3xNLS(SV40NLS):5&#39;-CCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTA-3&#39;. 
	NOTE: The 3xNLS at the 3&#39; end of Tau is cloned into the pCMV-Tau plasmid for mammalian expression exploiting the XhoI and BamHI restriction sites into the multicloning site (MCS).</li>
	<li>Generate Tau-NES construct by cloning by restriction enzyme digestion in frame at the 3&#39; end of Tau sequence 0N4R (383aa) the NES sequence: 5&#39;-AGTGAGCTGCAGAACAAGCTGGAAGAGTTGGATCTGGACTCGTACAA-3&#39;. 
	NOTE: The NES at the 3&#39; end of Tau is cloned into the pCMV-Tau plasmid for mammalian expression exploiting the EcoRI and BamHI restriction sites into the MCS.</li>
	<li>Transform DH5alpha E.coli strain with 100 ng of DNA from step 3.1 or 3.2 and plate cells on LB-Agar plates with 100 mg/mL ampicillin. Let grow overnight at 37 &#176;C.</li>
	<li>Pick a single colony and spike the cells into 5 mL of LB with ampicillin. Let the cells grow at 37 &#176;C in agitation overnight.</li>
	<li>Extract plasmid with a DNA miniprep (Table of Materials) and sequence to verify the constructs.</li>
	<li>Transform DH5alpha E. coli strain with the sequence verified constructs and plate cells on LB-Agar plates with ampicillin. Let grow overnight at 37 &#176;C.</li>
	<li>Pick a single colony and spike the cells into 5 mL of LB with ampicillin. Let the cells grow at 37 &#176;C in agitation for 2 h.</li>
	<li>Put the cells from step 3.7 in 200 mL of LB with ampicillin. Let grow overnight at 37 &#176;C.</li>
	<li>Pellet the cells at 3,500 x g for 10 min at 4 &#176;C.</li>
	<li>Extract plasmid with a DNA maxiprep (Table of Materials).</li>
</ol>
4. Cell Transfection
<ol>
	<li>Seed 400,000 cells from step 1.1 in 6-well plates or 20,000 cells in 8-well chamber slides. Plate four samples: control cells to be transfected with an empty vector, cells to be transfected with untagged Tau, cells to be transfected with Tau-NLS and cells to be transfected with Tau-NES.</li>
	<li>The day after seeding transfect 400 ng of DNA for each well using the cationic lipids (Table of Materials) in 6-well plates or 200 ng of DNA for each well for 8-well chamber slides, according to the manufacturer&#39;s instructions.
	<ol>
		<li>Incubate the DNA and the cationic lipids separately in 250 &#181;L (for 6-well plates) or 25 &#181;L (for 8-well chamber slides) of reduced serum medium for 5 min at RT. Then combine them to generate the DNA-lipid complex and incubate for 20 min.</li>
		<li>Replace the culture medium with 2 mL (for 6-well plates) or 250 &#181;L (for 8-well chamber slides) of fresh complete culture medium. Add the DNA-lipid complex to the cells and incubate at 37 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Alternatively, transfect DNA with the cationic reagent polyethylenimine (PEI).
	<ol>
		<li>Mix 2 &#181;g of DNA and 6 &#181;L of PEI with 200 &#181;L of complete culture medium (for each well in 6-well plates), or 1 &#181;g of DNA and 3 &#181;L of PEI with 100 &#181;L of complete culture medium (for each well in 8-well chamber slides), vortex and incubate for 10 min at RT.</li>
		<li>Add the mix to the cells and add 1.8 mL of complete culture medium per well in 6-well plates or 150 &#181;L of complete culture medium per well in 8-well chamber slides to reach plating volume.</li>
	</ol>
	</li>
	<li>Change the medium the day after transfection and add the differentiation media as described in step 2.2.</li>
</ol>
5. Immunofluorescence
<ol>
	<li>Remove the culture medium and rinse cells with 1x PBS. Fix cells with 100% ice cold methanol for 3 min without shaking. Remove the fixing solution and wash briefly with 1x PBS.</li>
	<li>Permeabilize with 0.1% non-ionic surfactant in 1x PBS for 5 min at room temperature (RT). Briefly, wash with 1x PBS, 3 times.</li>
	<li>Incubate cells with blocking buffer (0.1% Tween 20 and 1% BSA in PBS) for 30 min at RT on an orbital shaker.</li>
	<li>Incubate with appropriate primary antibodies (e.g., mouse monoclonal anti-Tau13 antibody) diluted 1:500 in blocking buffer overnight at 4 &#176;C on an orbital shaker. Remove the antibody solution and wash, briefly, with 1x PBS.</li>
	<li>Incubate with secondary antibodies conjugated to fluorophore (e.g., goat anti-mouse antibodies conjugated to Alexa Fluor 633) diluted 1:500 in blocking buffer for 1 h at RT. Remove the antibody solution and wash briefly with 1x PBS 3 times.</li>
	<li>To stain nuclei, incubate with DAPI diluted 1:20,000 in blocking buffer for 10 min at RT. Wash with 1x PBS 3 times. Mount coverslips on a slide using antifade mounting medium.</li>
</ol>
6. Western Blot
<ol>
	<li>To collect the cell pellet from step 4.4, remove the medium, and wash cells with PBS. Incubate with 500 &#181;L of 0.1% trypsin for 4 min at 37 &#176;C. Add an equal volume of complete medium and resuspend cells.</li>
	<li>Collect cells in a tube and centrifuge at 500 x g for 5 min. At the end of centrifugation carefully remove the supernatant. Add 1 mL of PBS, centrifuge at 500 x g for 5 min and carefully remove the supernatant. Store cell pellets on ice for immediate use or freeze at -80 &#176;C for long-term storage.</li>
	<li>For total protein extracts, incubate the cell pellet for 30 min on ice in lysis buffer (20 mM Tris-HCl pH 8, 20 mM NaCl, 10% glycerol, 1% octylphenoxy poly(ethyleneoxy)ethanol, branched (Table of Materials), 10 mM EDTA) supplemented with protease and phosphatase inhibitors. According to the abundance of the pellet, use 50 &#181;L to 100 &#181;L of lysis buffer.
	<ol>
		<li>Centrifuge the extract at 16,000 x g for 15 min. Collect the supernatant and quantify the protein concentration by any standard quantification assay. Prepare the protein samples for the SDS-PAGE by mixing 20 &#181;g of proteins with 5 &#181;L of 4x Laemmli buffer in a total volume of 20 &#181;L and boil at 100 &#176;C for 5 min. 
		NOTE: The sample can be stored at -20 &#176;C.</li>
	</ol>
	</li>
	<li>For subcellular fractionations, resuspend cells from step 6.2 in complete medium, and collect 1 x 106 cells per each sample. Centrifuge at 500 x g for 10 min to obtain cell pellets for the following steps.
	<ol>
		<li>To isolate subcellular compartments, fractionate according to kit specifications. To isolate each fraction, incubate the cell pellet from step 6.4 with the corresponding buffer, centrifuge, collect the supernatant and add the next buffer to the pellet as described in 6.4.1.1-6.4.1.5. Add in order cytoplasmic extraction buffer, membrane extraction buffer, nuclear extraction buffer, nuclear extraction buffer supplemented with 5 mM CaCl2 and 3 U/&#181;L micrococcal nuclease and cytoskeletal extraction buffer. 
		NOTE: All buffers must be supplemented with protease inhibitors. Scale buffer volumes according to the volume of the cell pellet.
		<ol>
			<li>To isolate the cytosolic fraction, incubate cell pellets in 100 &#181;L of ice-cold cytoplasmic extraction buffer supplemented with protease inhibitors at 4 &#176;C with gentle mixing for 10 min. Centrifuge at 500 x g at 4 &#176;C for 5 min and transfer the supernatant to pre-chilled tubes.</li>
			<li>Add 100 &#181;L of ice-cold membrane extraction buffer supplemented with protease inhibitors to the pellet from step 6.4.1.1, and incubate at 4 &#176;C with gentle mixing for 10 min. Centrifuge at 3,000 x g at 4 &#176;C for 5 min and collect the supernatant.</li>
			<li>For the soluble nuclear fraction, add 50 &#181;L of nuclear extraction buffer supplemented with protease inhibitors to the pellet from step 6.4.1.2, and vortex. Incubate at 4 &#176;C for 30 min, centrifuge at 5,000 x g at 4 &#176;C for 5 min, and collect the supernatant.</li>
			<li>For the insoluble nuclear fraction, add 50 &#181;L of nuclear extraction buffer supplemented with protease inhibitors, CaCl2 and micrococcal nuclease to the pellet from step 6.4.1.3, and vortex. Incubate at 37 &#176;C for 5 min, and then vortex again. Centrifuge at 16,000 x g at RT for 5 min and collect the supernatant.</li>
			<li>For the cytoskeletal fraction, add 50 &#181;L of cytoskeletal extraction buffer supplemented with protease inhibitors to the pellet from step 6.4.1.4, and vortex. Incubate 10 min at RT. Centrifuge the tube at 16,000 x g for 5 min, collect the supernatant and discard the pellet. 
			NOTE: Scale buffer volumes according to the cell volume, as indicated in the kit protocol. Refer to the kit protocol for further details on incubation and centrifugation time and temperature22,23,24,25,26,27,28,29. Alternatively, use any standard methods30 that, by using detergents and by increasing ionic strength and centrifugation speed, separates the cytosolic, the membrane-bound, the cytoskeletal and the nuclear fractions. Separate the soluble nuclear fraction and the insoluble nuclear fraction by exploiting standard nuclear extraction buffers. The sample can be stored at -20 &#176;C.</li>
		</ol>
		</li>
		<li>For the SDS-PAGE, add 7 &#181;L of 4x Laemmli buffer to 20 &#181;L of subcellular fractions obtained from steps 6.4.1.1&#8722;6.4.1.5, boil at 100 &#176;C for 5 min.</li>
	</ol>
	</li>
	<li>Load samples on an acrylamide gel and perform electrophoresis at a constant voltage of 120 V. Transfer proteins to nitrocellulose membrane at 250 mA for 90 min.</li>
	<li>Check proper protein gel electrophoresis and successful blotting by incubating the membrane for 5 min in Ponceau staining solution. Rinse the membrane in distilled water until the background is clean. Remove the stain by continued washing with Tris buffered saline with Tween 20 (TBST) for 10 min on a shaker.</li>
	<li>Incubate the membrane with blocking solution (5% milk in TBST) for 1 h at RT on shaker. Wash 3 times with TBST for 5 min.</li>
	<li>Hybridize the membrane with the primary antibody in blocking solution (1% milk in TBST) overnight at 4 &#176;C. Wash 3 times with TBST for 5 min.</li>
	<li>Hybridize the membrane with the HRP-conjugated secondary antibody in blocking solution for 1 h at RT. Wash 3 times with TBST for 5 min.</li>
	<li>Detect the protein band using chemiluminescence. Quantify the intensity of Western Blot bands by ImageJ. Normalize protein expression onto the product of a housekeeping gene: histone H2B for the nuclear soluble and insoluble fraction, GAPDH for the cytoplasmic fraction and for total extracts.</li>
</ol>

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U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Which+Medium+and+Ingredients+Provide+Better+Morphological+Differentiation+of+SH-SY5Y+Cells?.">Which Medium and Ingredients Provide Better Morphological Differentiation of SH-SY5Y Cells?.</a> Proceedings. 2 (25), 1577 (2018).</li><li>Forster, J. I., 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=Characterization+of+differentiated+SH-SY5Y+as+neuronal+screening+model+reveals+increased+oxidative+vulnerability.">Characterization of differentiated SH-SY5Y as neuronal screening model reveals increased oxidative vulnerability.</a> Journal of Biomolecular Screening. 21 (5), 496-509 (2016).</li><li>Dwane, S., Durack, E., Kiely, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimising+parameters+for+the+differentiation+of+SH-SY5Y+cells+to+study+cell+adhesion+and+cell+migration.">Optimising parameters for the differentiation of SH-SY5Y cells to study cell adhesion and cell migration.</a> BMC Research Notes. 6 (1), 1 (2013).</li><li>Encinas, M., 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=Sequential+Treatment+of+SH-SY5Y+Cells+with+Retinoic+Acid+and+Brain-Derived+Neurotrophic+Factor+Gives+Rise+to+Fully+Differentiated,+Neurotrophic+Factor-Dependent,+Human+Neuron-Like+Cells.">Sequential Treatment of SH-SY5Y Cells with Retinoic Acid and Brain-Derived Neurotrophic Factor Gives Rise to Fully Differentiated, Neurotrophic Factor-Dependent, Human Neuron-Like Cells.</a> Journal of Neurochemistry. 75 (3), 991-1003 (2002).</li></ol>

The authors have nothing to disclose.

We describe a method to measure the impact of nuclear Tau protein on gene expression. With this protocol the contribution of cytoplasmic Tau is strongly limited. Critical steps of this protocol are the following: the differentiation of human neuroblastoma SH-SY5Y cells, the subcellular fractionation and the localization of Tau protein in the nuclear compartment.
First, as shown in the representative results section, the differentiation of SH-SY5Y cells by adding RA and BDNF is crucial to obtain a good preparation of neuron-like cells in culture. The density of cells seeded is particularly important since a lower density might impact cell proliferation. Moreover, for experiments that need a high number of cells, like cellular fractionation and Western blot, it is important to note that the BDNF differentiation step blocks the cellular proliferation to allow the terminal differentiation, thus limiting the number of cells in culture. Alternative differentiation protocols use only RA or NGF instead of BDNF. However, while adding BDNF after RA allows to reach a better morphological differentiation32,33, NGF induces a weaker neurite outgrowth in SH-SY5Y cells34. Moreover, it has been extensively demonstrated that the combination of RA and BDNF allows to obtain a homogeneous neuronal population with expression of neuronal markers and decreased proliferation35. For this reason, the differentiation protocol exploited here combines RA and BDNF.
However, the procedure reported to dissect the role of Tau in different subcellular compartments can be used also for undifferentiated cells or for different cell types.
The subcellular fractionation is a very critical step andit is crucial to have enough starting material: a commercial kit requires only 1 x 106 cells, whereas other procedures may need a much higher starting quantity. Moreover, the use of a kit with standard buffers and steps guarantees the reproducibility of the experiment that is unavoidable and essential. However, since the composition of buffers is often proprietary, they might contain detergents which may alter the function of the protein of interest and it might be difficult to optimize the isolation of the fractions. Moreover, even in the best condition, there might be a 10-15% of contamination between fractions. A poor yield from each fraction could be overcome by increasing the incubation time in extraction buffers of specific fractions.
Since the functions of nuclear Tau have gained significant interest in recent years, it is particularly important to provide a reliable method to dissect the function of Tau in different cellular compartments. Coupling the subcellular fractionation, with the expression of Tau constructs specifically directed or excluded from the nucleus, allows one to finely tune the amount of Tau in different compartments.
A critical step in this part of the protocol is the cloning of Tau tagged with nuclear localization signal or with the nuclear export signal. The efficiency of the NLS is guaranteed by the presence of a 3XNLS consensus sequence from the SV40 virus. The nuclear translocation of the protein can be easily checked by immunofluorescence, and the lack of signal into the nucleus might be due to an incorrect cloning or to an inefficient transfection. On the contrary, the nuclear export is guaranteed by the NES consensus sequence. In this case, the immunofluorescence allows checking of the export of Tau from the nucleus. However, a weak nuclear signal is not to be excluded since Tau-NES protein enters the nucleus and then, due to the NES sequence, it is exported into the cytosol.
Up to now, the function of nuclear Tau has been studied only by correlative approaches that do not assure its direct involvement. The protocol here described, provide the first approach allowing to clearly discriminate the specific function of Tau into the nuclear compartment. As previously demonstrated, the endogenous Tau does not affect the results obtained by this protocol. Indeed, the same experiment performed in non-neuronal cells that do not express endogenous Tau, leads to VGluT1 altered expression. We applied this protocol to study the expression of disease-related genes31. Anyhow, it could be exploited also to investigate other nuclear Tau functions, such as the involvement on DNA damage, the interaction with nuclear cofactors or with the chromatin.

The functions of Tau protein in the nucleus have garnered significant interest in recent years, as it has been shown to be closely associated with nucleic acids1,2,3,4,5,6. Indeed, a recent genome-wide study demonstrated that Tau binds genic and intergenic DNA sequences in vivo7. A role in nucleolar organization has been suggested8,9,10,11. Moreover, Tau has been proposed to be involved in DNA protection from oxidative and hyperthermic stress5,10,12,13, whereas mutated Tau has been linked to chromosome instability and aneuploidy14,15,16.
Until now, the challenges in studying the functions of Tau in the nuclear compartment remained almost unsolved due to the difficulties in dissecting the specific contribution of nuclear Tau from the contribution of cytoplasmic Tau. Moreover, the functions attributed to Tau molecules in the nuclear compartment, up to now, are only correlative because they lack an unequivocal demonstration of the direct involvement of nuclear Tau proteins. Indeed, the involvement of Tau in the mobility of retrotransposons or in the rRNA transcription or in DNA protection11,12,17,18,19 might be also explained by the contribution of cytoplasmic Tau or by the effect of other downstream factors not related to nuclear Tau.
Here, we provide a method that can solve this issue by exploiting a classical procedure to isolate the nuclear compartment combined with the use of Tau constructs 0N4R tagged with nuclear localization (NLS) or nuclear export signals (NES). This approach eliminates the complex issues related to possible artefacts due to the spillover of Tau molecules from the cytoplasmic compartment. Moreover, Tau-NLS and Tau-NES constructs induce the enrichment or the exclusion of Tau molecules from the nuclear compartment, respectively, providing positive and negative controls for the involvement of nuclear Tau molecules in a specific function. The protocol is technically easy and it is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types, differentiated or not, such as cancer cells that reactivate Tau expression20,21. Moreover, it might be applied also to other proteins that are present in both the cytoplasm and the nucleus in order to dissect biological functions related to different compartments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 633 goat anti-mouse IgG</td>
			<td>Life Technologies</td>
			<td>A21050</td>
			<td>IF 1:500</td>
		</tr>
		<tr>
			<td>anti Actin Antibody</td>
			<td>BETHYL LABORATORIE</td>
			<td>A300-485A</td>
			<td>anti-rabbit WB 1:10000</td>
		</tr>
		<tr>
			<td>anti GAPDH Antibody</td>
			<td>Fitzgerald Industries International</td>
			<td>10R-G109a</td>
			<td>anti-mouse WB 1:10000</td>
		</tr>
		<tr>
			<td>anti H2B Antibody</td>
			<td>Abcam</td>
			<td>ab1790</td>
			<td>anti-rabbit WB 1:15000</td>
		</tr>
		<tr>
			<td>anti Tau-13 Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-21796</td>
			<td>anti-mouse WB 1:1000; IF 1:500</td>
		</tr>
		<tr>
			<td>anti Tubulin alpha Antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>PA5-16891</td>
			<td>anti-mouse WB 1:5000</td>
		</tr>
		<tr>
			<td>anti VGluT1 Antibody</td>
			<td>Sigma-Aldrich</td>
			<td>AMAb91041</td>
			<td>anti-mouse WB 1:500</td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Euroclone</td>
			<td>EMPO14500</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Alomone Labs</td>
			<td>B-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Blotting-Grade Blocker</td>
			<td>Biorad</td>
			<td>1706404</td>
			<td>Non-fat dry milk</td>
		</tr>
		<tr>
			<td>BOVIN SERUM ALBUMIN</td>
			<td>Sigma-Aldrich</td>
			<td>A4503-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Mini</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td>protease inhibitor</td>
		</tr>
		<tr>
			<td>Criterion TGX 4-20% Stain Free, 10 well</td>
			<td>Biorad</td>
			<td>5678093</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>62247</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>GIBCO</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium Low Glucose</td>
			<td>Euroclone</td>
			<td>ECM0060L</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>0390-100ml</td>
			<td>pH=8 0.5M</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum</td>
			<td>Euroclone</td>
			<td>EC50182L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG-HPR</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
			<td>WB 1:1000</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG-HPR</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2004</td>
			<td>WB 1:1000</td>
		</tr>
		<tr>
			<td>IGEPAL CA-630</td>
			<td>Sigma-Aldrich</td>
			<td>I8896-50ml</td>
			<td>Octylphenoxy poly(ethyleneoxy)ethanol</td>
		</tr>
		<tr>
			<td>Immobilon Western</td>
			<td>MERCK</td>
			<td>WBKLS0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab-Tech Chamber slide 8 well glass slide</td>
			<td>nunc</td>
			<td>177402</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Euroclone</td>
			<td>ECB3000D</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 transfection reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>12566014</td>
			<td>cationic lipid</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>322415-6X1L</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM reduced serum medium</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Sigma-Aldrich</td>
			<td>40,872-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>10,000 U/ml, 100ml</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (Dulbecco A)</td>
			<td>OXOID</td>
			<td>BR0014G</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosStop</td>
			<td>Roche</td>
			<td>4906837001</td>
			<td>phosphatase inhibitor</td>
		</tr>
		<tr>
			<td>QIAGEN Plasmid Maxi Kit</td>
			<td>Qiagen</td>
			<td>12163</td>
			<td>Step 3.10</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>R2625-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Subcellular Protein Fractionation Kit for cultured cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>78840</td>
			<td></td>
		</tr>
		<tr>
			<td>Supported Nitrocellulose membrane</td>
			<td>Biorad</td>
			<td>1620097</td>
			<td></td>
		</tr>
		<tr>
			<td>TC-Plate 6well</td>
			<td>SARSTEDT</td>
			<td>833,920</td>
			<td></td>
		</tr>
		<tr>
			<td>TCS SP2 laser scanning confocal microscope</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton x-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-500ml</td>
			<td>Non-ionic surfactant</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15400054</td>
			<td>0.50%</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD antifade mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard Plus SV Minipreps DNA Purification Systems</td>
			<td>Promega</td>
			<td>A1330</td>
			<td>Step 3.5</td>
		</tr>
	</tbody>
</table>

modulation of tau subcellular localization as a tool to investigate the expression of disease-related genes

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, recent evidence indicated that Tau is present also in other subcellular compartments including the nucleus where it is implicated in DNA protection, in rRNA transcription, in the mobility of retrotransposons and in the structural organization of the nucleolus. We have recently demonstrated that nuclear Tau is involved in the expression of the VGluT1 gene, suggesting a molecular mechanism that could explain the pathological increase of glutamate release in the early stages of Alzheimer&#39;s disease. Until recently, the involvement of nuclear Tau in modulating the expression of target genes has been relatively uncertain and ambiguous due to technical limitations that prevented the exclusion of the contribution of cytoplasmic Tau or the effect of other downstream factors not related to nuclear Tau. To overcome this uncertainty, we developed a method to study the expression of target genes specifically modulated by the nuclear Tau protein. We employed a protocol that couples the use of localization signals and the subcellular fractionation, allowing the exclusion of the interference from the cytoplasmic Tau molecules. Most notably, the protocol is easy and is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types and cellular conditions.

The strategy used to dissect the impact of nuclear Tau in gene expression avoiding the contribution of cytoplasmic Tau proteins has been depicted in Figure 1. Briefly, Tau proteins tagged with NLS or NES are accumulated in or excluded from the nuclear compartment, respectively. The functional effect of this unbalance is the alteration of the gene expression measured as the product of the VGluT1 gene.
Following the protocol description, SH-SY5Y cells were treated with RA for 5 days and then with BDNF for 3 days in order to obtain post-mitotic neuron-like cells (Figure 2). In the absence of RA and BDNF, undifferentiated SH-SY5Y cells assume a rounder morphology and form cell clumps. As expected, starting the differentiation protocol, clumps unwind and cells spread out neurites; at the end of differentiation, cells are uniformly distributed and interconnected via a network of branched neurites.
The day after seeding, cells have been transfected with Tau-NLS or Tau-NES plasmids (section 4.2) with cationic lipids. For cells expressing Tau-NLS or Tau-NES constructs, Tau subcellular localization can be detected by immunofluorescence with anti-Tau antibodies. Depending on the efficiency of transfection, cells display a strong nuclear staining merging with the DAPI signal or a cytoplasmic staining with empty nuclei if they are successfully transfected with Tau-NLS or Tau-NES, respectively (Figure 3). The lack of these specific signals indicates an inefficient transfection.
To analyse the proteins enriched in different subcellular compartment, cells were collected and counted in order to process equal amounts of cells per sample. Any standard fractionation method that exploits increasing detergent and ionic strength and increasing centrifugation speed can be used to separate the cellular compartments from one another and thus isolate the cytosolic, the membrane-bound, the cytoskeletal and the nuclear fractions.
Once the nuclei have been isolated, the nuclear soluble fraction and the chromatin bound fraction were separated by adding 3 U/&#181;L of micrococcal nuclease and 5 mM CaCl2.
For Western blot analysis, equal volumes of cytoplasmic and membrane fractions and half volumes of the other fractions have been loaded on a gradient precast acrylamide gel, to correct for the different amount of buffer added at each step.
To verify the efficient separation of different fractions, the Western blot exploiting the following antibodies has been performed: anti-GADPH (present in all fractions except the cytoskeleton and particularly enriched in the cytoplasmic fraction); anti-actin (particularly enriched in the cytoskeletal fraction); anti-tubulin (particularly enriched in Cytoplasmic and cytoskeletal fractions); anti-H2B (enriched in the nuclear fractions) (Figure 4).
An enrichment of these markers in different subcellular fractions indicates that the fractionation is not well performed. It must be noted that any protocol for subcellular fractionation might present a 10-15% of contamination between fractions.
Once verified the successful fractionation of the sample, the Western blot has been performed to check the signal of Tau in the nuclear compartment and the VGluT1 signal in the total extract (Figure 5). While untagged Tau is detectable in all fractions, Tau-NLS is strongly enriched in the nuclear compartment and it is poorly detectable in the cytoplasmic fraction. On the contrary, Tau-NES is enriched in the cytoplasmic fraction and it is less detectable in the nuclear fraction. The presence of a small amount of Tau-NES into the soluble nuclear fraction has to be expected since, like the endogenous Tau, it is translocated into the nucleus and once into the nuclear compartment the nuclear export signal allows its translocation to the cytoplasm. The detection of a different enrichment for these two fusion proteins might indicate a problem in the efficiency of transfection or in the cloning of constructs or in fractionation.
Quantitative analysis of Western blot can be done using ImageJ. Values are normalized for the housekeeping gene specific for each fraction (GAPDH for cytoplasmic fraction; histone H2B for soluble nuclear and chromatin-bound fractions).
The graph in Figure 5B reports the ratio of Tau in the soluble nuclear fraction and cytoplasmic fraction to highlight that Tau-NLS is highly enriched in the soluble nuclear fraction (SNF) while Tau-NES is decreased. Moreover, Tau-NLS is enriched in the chromatin-bound fraction (CBF) with respect to the cytoplasmic fraction (CF) while Tau-NES is decreased. SNF/CF = 1 and CBF/CF = 1 correspond to Tau ratio in control cells. The endogenous Tau is weakly detectable in all fractions as expected. The graph in Figure 5C reports the quantification of VGluT1 expression in the total extracts of samples expressing different amount of nuclear Tau. In cells expressing Tau-NES, VGluT1 expression is comparable to the baseline expression in control cells. On the contrary, in cells expressing untagged Tau or Tau-NLS, the expression of VGluT1 is more than doubled.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59988/59988fig1a.jpg" /> 
Figure 1: Graphical representation of the strategy used to allow a nuclear or a cytoplasmic accumulation of Tau. Tau-NLS is accumulated in the nuclear compartment while Tau-NES is excluded. The experimental readout is the modulation of the VGluT1 expression. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59988/59988fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59988/59988fig2a.jpg" /> 
Figure 2: Representative undifferentiated and differentiated cell culture. Image of undifferentiated SH-SY5Y (left), cells differentiated by RA (middle) and differentiated by RA and BDNF (right). Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59988/59988fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59988/59988fig3a.jpg" /> 
Figure 3: Representative image of Tau subcellular enrichment by immunofluorescence. Image of cells untransfected or expressing untagged Tau, Tau-NES or Tau-NLS constructs. Tau signal has been obtained by immunofluorescence (red), nuclei signal has been obtained by DAPI staining (blue), merged images are reported. Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59988/59988fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59988/59988fig4a.jpg" /> 
Figure 4: Representative detection of proteins enriched in subcellular fractions by Western blot. Western blot of subcellular fractions from SH-SY5Y cells. CF = cytoplasmic fraction; MF = membrane fraction; SNF = soluble nuclear fraction; CBF = chromatin bound fraction; CKF = cytoskeletal fraction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59988/59988fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59988/59988fig5a.jpg" /> 
Figure 5: Representative detection of nuclear Tau and VGluT1 proteins. (A) Western blot of Tau protein detected in the nuclear and cytoplasmic fractions. (B) The graph reports the ratio of Tau in the nuclear fractions and cytoplasmic fraction. The values have been normalized on the endogenous Tau. SNF/CF = 1 and CBF/CF = 1 corresponds to endogenous Tau ratio in control cells. (C) Western blot of VGluT1 protein. (D) The graph reports the quantification of VGluT1 expression in the total extracts of samples expressing different amount of nuclear Tau. Kruskal-Wallis ANOVA and Mann-Whitney test; *** p &#60; 0.001, **** p &#60; 0.0001, n.s. p &#62; 0.05. All results are shown as mean &#177; SEM from at least three independent experiments. This representative figure has been modified from Siano et al.31. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59988/59988fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes at JoVE.com

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Tau is a neuronal protein present both in the cytoplasm, where it binds microtubules, and in the nucleus, where it exerts unconventional functions including the modulation of Alzheimer&#39;s disease-related genes. Here, we describe a method to investigate the function of nuclear Tau while excluding any interferences coming from cytoplasmic Tau.

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, ...

modulation-tau-subcellular-localization-as-tool-to-investigate

Nanyang Technological University

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JoVE Journal

Genetics

6.2K Views.  Scuola Normale Superiore. Tau is a neuronal protein present both in the cytoplasm, where it binds microtubules, and in the nucleus, where it exerts unconventional functions including the modulation of Alzheimer's disease-related genes. Here, we describe a method to investigate the function of nuclear Tau while excluding any interferences coming from cytoplasmic Tau.

Video: Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on animal physiology and behavior. Recently, we showed that modulation of longevity in the nematode Caenorhabditis elegans by food abundance is more complex than previously recognized. The responsiveness of the lifespan to changes in food level is determined by specific genes that act by controlling information processing within a neural circuit. Our framework combines genetic analysis, high-throughput quantitative imaging and information theory. Here, we describe how these techniques can be used to characterize any gene that has a physiological relevance to broad-range dietary restriction. Specifically, this workflow is designed to reveal how a gene of interest regulates lifespan under broad-range dietary restriction; then to establish how the expression of the gene varies with food level; and finally, to provide an unbiased quantification of the amount of information conveyed by gene expression about food abundance in the environment. When several genes are examined simultaneously under the context of a neural circuit, this workflow can uncover the coding strategy employed by the circuit.

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Here, we present a framework to relate broad-range dietary restriction to gene expression and lifespan. We describe protocols for broad-range dietary restriction and for quantitative imaging of gene expression under this paradigm. We further outline computational analyses to reveal underlying information processing features of the genetic circuits involved in food-sensing.

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on ...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a polymorphic cluster of genes on chromosome 19. Their best-characterized ligands are the human leukocyte antigen (HLA) molecules that are encoded within the major histocompatibility complex (MHC) locus on chromosome 6. There is substantial evidence that they play a significant role in immunity, reproduction, and transplantation, making it crucial to have techniques that can accurately genotype them. However, high-sequence homology, as well as allelic and copy number variation, make it difficult to design methods that can accurately and efficiently genotype all KIR genes. Traditional methods are usually limited in the resolution of data obtained, throughput, cost-effectiveness, and the time taken for setting up and running the experiments. We describe a method called quantitative KIR semi-automated typing (qKAT), which is a high-throughput multiplex real-time polymerase chain reaction method that can determine the gene copy numbers for all genes in the KIR locus. qKAT is a simple high-throughput method that can provide high-resolution KIR copy number data, which can be further used to infer the variations in the structurally polymorphic haplotypes that encompass them. This copy number and haplotype data can be beneficial for studies on large-scale disease associations, population genetics, as well as investigations on expression and functional interactions between KIR and HLA.

Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies.

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

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Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Controllable Ion Channel Expression through Inducible Transient Transfection

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression