Name: modulation of tau subcellular localization as a tool to investigate the expression of disease-related genes
Uploaded: 2019-12-20T12:00:00
Description: 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

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 cel...

<ol>
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Q., Bartlett, P. F., G&#246;tz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+the+microtubule-associated+protein+tau+in+the+nucleus.">Visualizing the microtubule-associated protein tau in the nucleus.</a> Science China Life Sciences. 57 (4), 422-431 (2014).</li><li>Sultan, A., 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=Nuclear+Tau,+a+key+player+in+neuronal+DNA+protection.">Nuclear Tau, a key player in neuronal DNA protection.</a> Journal of Biological Chemistry. 286 (6), 4566-4575 (2011).</li><li>Sj&#246;berg, M. K., Shestakova, E., Mansuroglu, Z., Maccioni, R. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+specific+tau+exons+in+normal+and+tumoral+pancreatic+acinar+cells.">Expression of specific tau exons in normal and tumoral pancreatic acinar cells.</a> Journal of Cell Science. 111 (1), 1419-1432 (1998).</li><li>Liao, A., 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=Therapeutic+efficacy+of+FTY720+in+a+rat+model+of+NK-cell+leukemia.">Therapeutic efficacy of FTY720 in a rat model of NK-cell leukemia.</a> Blood. 118 (10), 2793-2800 (2011).</li><li>Cascio, S., Zhang, L., Finn, O. 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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 obtai...

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.
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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, ...

This method can help answer key questions about the direct role of tau in the nuclear compartment while excluding any potential cytoplasmic tau contamination. The main advantage of this technique is that it is broadly applicable to the study on the nuclear function of tau in other cell types and under different cellular conditions. Demonstrating the procedure will be Giacomo Siano, a post-doc from my laboratory.Begin by plating four times 10 to the five SH-SY5Y human neuroblastoma cell line cells to be transfected with the empty control vector, four times 10 to the five cells to be transfected with untagged tau, four times 10 to the five cells to be transfected with tau-NLS, and four times 10 to the five cells to be transfected with tau-NES into individual wells of a six-well plate. The day after plating, separately incubate 400 nanograms of DNA and the appropriate volume of cationic lipids into one tube of 250 microliters of reduced serum per well. After five minutes at room temperature, combine each vector with one volume of cationic lipid and incubate the DNA lipid complexes for 20 minutes at room temperature.At the end of the incubation, replace the supernatants in each well with two milliliters of fresh complete culture medium and transfect each well with the appropriate DNA lipid complex solution for an overnight incubation at 37 degrees Celsius. The next day, replace the supernatants with fresh differentiation medium. For Western Blot analysis, wash the transfected and differentiated cells in each well with PBS and incubate the cells with 500 microliters of 0.1%Trypsin per well for four minutes at 37 degrees Celsius.When the cells have detached, stop the reaction with an equal volume of complete medium and transfer the cell suspension from each well into individual tubes for centrifugation. After carefully removing the supernatants, resuspend the pellets in one milliliter of PBS per tube for a second centrifugation and remove the supernatants again. For total protein extracts, incubate the pellets for 30 minutes on ice in 50 to 100 microliters of lysis buffer supplemented with protease and phosphatase inhibitors.At the end of the incubation, centrifuge the extract at 16, 000 times g for 15 minutes and quantify the protein concentration by any standard quantification assay. Then mix 20 micrograms of each protein sample with five microliters of 4X Laemmli buffer to a total volume of 20 microliters and boil the samples at 100 degrees Celsius for five minutes. For subcellular fractionation, resuspend the cells in complete medium for counting and spin down one times 10 to the six cells per sample.To isolate the cytosolic fraction, incubate the pellets in 100 microliters of ice cold cytoplasmic extraction buffer supplemented with protease inhibitors at four degrees Celsius with gentle mixing for 10 minutes. At the end of the incubation, collect the samples by centrifugation and transfer the supernatants into pre-chilled tubes. Add 100 microliters of ice cold membrane extraction buffer supplemented with protease inhibitors to the pellet for a 10 minute incubation at four degrees Celsius with gentle mixing.Then centrifuge the pellets for five minutes at four degrees Celsius and 3, 000 times g and collect the supernatants into new tubes. To collect the soluble nuclear fraction, add 50 microliters of nuclear extraction buffer supplemented with protease inhibitors to the pellets with vortexing before incubating the samples at four degrees Celsius for 30 minutes. At the end of the incubation, centrifuge the samples and collect the supernatants.To collect the insoluble nuclear fraction, add 50 microliters of nuclear extraction buffer supplemented with protease inhibitors, calcium chloride and micrococcal nuclease to the pellets with vortexing. Incubate the samples for five minutes at 37 degrees Celsius before vortexing and centrifuging. Then collect the supernatants.To collect the cytoskeletal fraction, add 50 microliters of cytoskeletal extraction buffer supplemented with protease inhibitors to the pellets with vortexing. After a 10 minute incubation at room temperature, centrifuge the samples and collect the supernatants. For SDS-PAGE analysis, add seven microliters of 4X Laemmli buffer to 20 microliters of each of the collected subcellular fraction samples and boil the samples at 100 degrees Celsius for five minutes.At the end of the incubation, load the samples onto an acrylamide gel and perform the electrophoresis at a constant voltage of 120 volts. Then transfer the proteins to a nitrocellulose membrane at 250 milliamps for 90 minutes. Incubate the membrane for five minutes in Ponceau staining solution to check for proper protein gel electrophoresis and a successful blotting.At the end of the incubation, rinse the membrane in distilled water until the background is clean and remove the stain with continuous washing with Tris-buffered saline supplemented with Tween 20 for 10 minutes on a shaker. Next, incubate the membrane with blocking solution for one hour at room temperature with shaking before washing three times with Tris-buffered saline plus Tween 20 for five minutes per wash. After the last wash, hybridize the membrane with the primary antibody of interest in blocking solution overnight at four degrees Celsius followed by three washes with Tris-buffered saline plus Tween 20 for five minutes.After the last wash, hybridize the membrane with an appropriate horseradish peroxidase conjugated secondary antibody in blocking solution for one hour at room temperature before washing the membrane with Tris-buffered saline plus Tween 20 three times for five minutes per wash. After the last wash, detect the protein band using chemiluminescence and quantify the intensity of Western Blot bands by ImageJ. In the absence of retinoic acid and brain-derived neurotrophic factor, undifferentiated SH-SY5Y cells assume a rounder morphology and form cell clumps.As expected, after five days of treatment with retinoic acid, the clumps unwind and the cells spread out. After three additional days of treatment with brain-derived neurotrophic factor, the cells appear uniformly distributed and interconnected via a network of branched neurites. After tau-NLS or tau-NES transfection, tau subcellular localization can be detected by immunofluorescence with anti-tau antibodies.Depending on the efficiency of the transfection, the cells display a strong nuclear staining merging with the DAPI signal after tau-NLS transfection or a cytoplasmic staining with the empty nuclei after tau-NES. Western Blot analysis reveals the enrichment of actin within the cytoskeletal fraction, tubulin within the cytoplasmic and cytoskeletal fractions, and H2B within the nuclear fractions. Western Blot analysis also confirms the expression of tagged and untagged tau within the nuclear compartment and vesicular glutamate transporter one expression in the total extract.Indeed, the ratio of tau in the soluble nuclear and cytoplasmic fractions highlights the fact that tau-NLS is highly enriched in the soluble nuclear fraction while tau-NES is decreased. Moreover, tau-NLS is enriched in the chromatin-bound fraction with respect to the cytoplasmic fraction while tau-NES is decreased. Be sure to carefully check the number of cells used for the fractionation of each sample and to verify the enrichment of the housekeeping genes in the proper fractions.After watching this video, you should be able to isolate cellular compartments and to evaluate the functional role of tau in the nucleus.

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

Research

JoVE Journal

Genetics

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 ...