S.L. was supported by a fellowship from the DFG (Graduate Research School 845). This work was supported by grants from the DFG (SFB 530/C1 &amp; FOR 967) and by HOMFOR.

1. Preparation of stock solutions
<ol>
<li>Prepare calcium-free buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.5 mM EGTA, 10 mM glucose in 10 mM HEPES-KOH, (pH 7.35)).</li>
<li>Solubilize FURA-2 AM in DMSO to obtain an 1 mM stock solution. Mix using a vortex until the solution is homogenously light yellow. Protect cup from light. Dilute the FURA-2 AM stock solution into 1 ml Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin to a final concentration of 4 &mu;M for loading of HeLa cells.</li>
<li>Prepare a 12.5 mM puromycin stock solution in distilled water (pH 7.0). Store aliquots at -20&deg;C. Dilute the 12.5 mM puromycin stock solution in the calcium-free buffer to a final concentration of 500 &mu;M puromycin.</li>
<li>Dissolve thapsigargin in DMSO to obtain a 1 mM Stock solution.</li>
<li>Solubilize 1 mg ophiobolin A in 20 &mu;l chloroform and, subsequently, mix with 25 &mu;l DMSO. The microcentrifuge tube remains open until chloroform is evaporated. Thus the final ophiobolin A concentration is 100 mM. Store aliquots at -20 &deg;C. Prior to use, dilute stock solutions into calcium-free buffer to a final concentration of 100 &mu;M. Thus, final DMSO concentration for live cell imaging does not exceed 0.1%.</li>
<li>Dissolve trifluoperazine in DMSO to a concentration of 10 mM and store aliquots at -20 &deg;C. Dilute stock solutions into calcium-free buffer to a final concentration of 10 &mu;M prior to use. Thus final DMSO concentration for live cell imaging, like for ophiobolin A, does not exceed 0.1%.</li>
<li>Dissolve the siRNAs (SEC61A1 siRNA, SEC61A1-UTR siRNA and control siRNA) in RNase free water to prepare a 20 &mu;M stock solution. Mix using a vortex and observe solubilization by eye. Store 50 &mu;l aliquots at -20 &deg;C.</li>
</ol>
2. Gene silencing in HeLa cells
In order to study the contribution of a certain protein to ER Ca2+ efflux, the respective gene has to be efficiently silenced with two different siRNAs (Fig. 1). In addition, the effect of silencing has to be overcome by expression of the respective wild type gene. Typically we use siRNAs that are directed against the coding and non-coding (UTR) region, respectively, of the gene of interest. Employing UTR-directed siRNA provides a convenient way for complementation.
<ol>
	<li> Seed 5.2 x 105 HeLa cells (ATCC no. CCL-2) in a 6 cm culture dish with a 25 mm cover slip (pre-treated with 200 &mu;l of poly-L-lysine (1 mg/ml) for 1 h) in Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and incubate at 37&deg;C in a humidified environment with 5% CO2 (final volume 3.9 ml). </li>
<li>Transfect the cells with SEC61A1 siRNA, SEC61A1-UTR siRNA, or a negative control siRNA using HiPerFect Reagent according to the manufacturer&#x2019;s instructions (final concentration of siRNA: 20 nM). Briefly, prepare siRNAs freshly in a separate microcentrifuge tube prior to the actual transfection procedure: Add 20 &mu;l HiPerFect transfection reagent to 4 &mu;l of each siRNA (20 &mu;M) that is dissolved in 80 &mu;l of OptiMEM. This mix is gently vortexed and incubated at room temperature for 10 min. Add the siRNA mix (104 &mu;l) dropwise to the seeded 5.2 x 105 HeLa cells (3.9 ml).</li>
<li>After 24 h, change the medium (3.9 ml) and transfect the cells for a second time with the same siRNA mix (104 &mu;l).</li> 
<li>Evaluate silencing by Western blot analysis. It should be above 80%. Wash cells from the cell culture dish in phosphate-buffered saline (PBS) and harvest them in DMEM. Count the cells by employing the Countess Automated Cell Counter. Lyse the cells in cell lysis buffer (10 mM NaCl, 10 mM Tris-HCl, pH 8.0, 3 mM MgCl2, 0,5% NP40) supplemented with a protease-inhibitor cocktail (3 &mu;g/ml Pepstatin A, 3 &mu;g/ml Leupeptin, 3 &mu;g/ml Antipain, 3 &mu;g/ml Chymastatin) and mix with SDS-sample buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 10% Glycerol, 5% &beta;-mercaptoethanol, 0.01% bromphenol blue) to obtain a final concentration of 10.000 cells/&mu;l. Heat samples at 56 &deg;C for 15 min and, subsequently, add some glas beads and vortex for 20 min to fragment DNA. Separate 20 &mu;l of each sample by a Laemmli-type SDS-PAGE (typically 15% polyacrylamid for the separating gel). Transfer separated proteins onto a PVDF membrane by electroblotting (&#x201C;wet-blot&#x201D;) at constant 400 mA for 3 h or overnight in transfer buffer (100 mM Glycin, 12.5 mM Tris-HCl). Afterwards, block with 5% (w/v) low fat dried milk in PBS for 30 min at room temperature and then expose the blot to the primary antibody directed against the C-terminus of Sec61&alpha; from rabbit and an anti-&beta;-actin-antibody from mouse. Wash the blot in PBS-TritonX-100 (0.05%) and twice in PBS for 5 min, respectively. Visualize the primary antibodies using ECL Plex goat-anti-rabbit IgG-Cy5- or ECL Plex goat-anti-mouse IgG-Cy3-conjugate and the Typhoon-Trio imaging system in combination with the Image Quant TL software 7.0. In the case of the SEC61A1 gene, the maximum silencing effect is typically seen 96 h after the first transfection. A representative experiment is shown in Fig. 2.</li>
</ol>
3. Complementation of HeLa cells
In order to rescue the phenotype of SEC61A1 silencing, the SEC61A1 cDNA was inserted into the multiple cloning site (MCS) of a pCDNA3-internal ribosomal entry site (IRES)-GFP-vector that contained the cytomegalovirus (CMV) promoter, the MCS, the IRES, plus the green fluoresecent protein (GFP) coding sequence. 
<ol>
<li>48 h after the first siRNA transfection, exchange the medium (3.9 ml) for a second time and transform the cells with either empty vector or SEC61A1 expression plasmid using Fugene HD according to the manufacturer&#x2019;s protocol (final ratio of vector to Fugene HD is 4 &mu;g vector to 16 &mu;l Fugene HD). Briefly, prepare plasmids freshly in a separate microcentrifuge tube prior to the transformation procedure: Add 16 &mu;l Fugene HD transformation reagent to 4 &mu;g of each plasmid that is dissolved in 80 &mu;l of OptiMEM. Gently vortex this mix and incubate at room temperature for 10 min. Add the plasmid mix (0.1 ml) dropwise to the seeded 5.2 x 105 HeLa cells (3.9 ml).</li>
<li>After 48 h of plasmid expression, subject culture dishes to fluorescence microscopy prior to calcium imaging and harvesting. If necessary replace DMEM by PBS for better fluorescence signals. Record fluorescence images on a fluorescence microscope equipped with cooled CCD camera. Transformation efficiency can be determined by dividing the number of cells displaying GFP fluorescence by cells counted in the brightfield mode and should be above 80%. A typical result is shown in Fig. 3.</li>
</ol>
4. Live cell calcium imaging
<ol>
<li>Prior to measurement, transfer the cover slip coated with transfected HeLa cells in a separate 3.5 cm culture dish. Load the cells with 4 &mu;M FURA-2 AM in 1 ml DMEM for 45 min at 25&deg;C in the dark11,12.</li> 
<li>Fix a 25 mm cover slip in the iMIC metal chamber and wash cells twice and incubate in a calcium-free buffer (each time 300 &mu;l).</li>
<li>Start collecting data on an iMIC microscope with polychrome V by alternately exciting at 340 nm and 380 nm and measuring the emitted fluorescence at 510 nm (Filter set: beamsplitter, 565 nm; emitter, 605/70 nm). Sample images containing 20-50 cells/frame every 3 s. Record FURA-2 signals as the ratios F340/F380, where F340 and F380 correspond to the background-subtracted fluorescence intensity at 340 and 380 nm, respectively.</li> 
<li>After 1 min, treat cells with puromycin (500 &mu;M) in calcium-free buffer or with the same buffer. Alternatively, treat cells with a small molecule inhibitor (such as 10 &mu;M trifluoperazine or 100 &mu;M ophibolin A) or with the calcium-free buffer.</li> 
<li>After ratiometric measurements are carried out for 2 or 10 min, add thapsigargin (1 &mu;M) and continue the measurements.</li> 
<li>Eventually, cytoslic [Ca2+] is estimated from ratio measurements by an established calibration method.2 Analyze data by Excel 2007 and Origin 6.1.</li>
</ol>
5. Representative Results: 
So far, we addressed the question of whether silencing of the SEC61A1 gene affects calcium (Ca2+) leakage from the ER (Fig. 1). The SEC61A1 gene was silenced by two different siRNAs in HeLa cells for 96 hours. While silencing hardly affected cell growth and viability, protein transport into the ER of semi-permeabilized cells was almost completely inhibited. Furthermore, the SEC61A1 silenced cells were severely affected with respect to Ca2+ leakage from the ER. The effect of gene silencing was reversed by expression of the SEC61A1 gene. Thus, Sec61 complexes that are present in the ER membrane of all nucleated cells form Ca2+ leak channels that can be expected to play a crucial role in Ca2+ homeostasis. However, the presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of Ca2+ from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM) binding motif in the cytosolic N-terminus of mammalian Sec61&alpha; that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. At the cellular level, two different CaM antagonists stimulated Ca2+ release from the ER in the presence but not in the absence of Sec61 channels (Figs 4 and 5). The latter was not observed when Sec61 channels were present that contained mutations in the IQ motif of Sec61&alpha;. Thus, Ca2+-CaM is involved in limiting Ca2+ leakage from the ER (Fig. 6).
<img src="/files/ftp_upload/2730/2730fig1.jpg" alt="Figure 1"/> Figure 1. Flow chart. See text for details.
<img src="/files/ftp_upload/2730/2730fig2.jpg" alt="Figure 2"/> Figure 2. Data analysis for silencing efficiency. Silencing was evaluated by Western-blot analysis using antibodies that were directed against Sec61&alpha; and &beta;-actin (loading control). The primary antibodies were visualized by using ECL Plex secondary antibodies and fluorescence imaging.
<img src="/files/ftp_upload/2730/2730fig3.jpg" alt="Figure 3"/> Figure 3. Data analysis for transfection efficiency. Images were recorded on a fluorescence microscope equipped with cooled CCD camera. Transformation efficiency can be determined by dividing the number of cells displaying GFP fluorescence by cells counted in the brightfield mode.
<img src="/files/ftp_upload/2730/2730fig4.jpg" alt="Figure 4"/> Figure 4. Screen shots from live cell calcium imaging of HeLa cells in the presence of control- or SEC61A1-siRNA and the presence or absence of the CaM-antagonist ophiobolin A. HeLa cells were treated with siRNA directed against SEC61A1 (B) or a negative control siRNA (A) for 96 h as indicated. These cells were loaded with the calcium indicator FURA-2 AM and incubated in a Ca2+ free buffer containing 0.5 mM EGTA, then buffer or ophiobolin A (Ophio A) in buffer was added and the incubation resumed. After 10 min, Ca2+ release was initiated by applying thapsigargin (TG) in the absence of external Ca2+ and the incubation was continued. Screen shots from the continuous calcium imaging were taken at the indicated times.
<img src="/files/ftp_upload/2730/2730fig5.jpg" alt="Figure 5"/> Figure 5. Data analysis for live cell calcium imaging experiments. (A) Kinetic and quantitative analysis of a series of experiments as depicted in Fig. 4A. Cytoslic [Ca2+] was estimated from ratio measurements by an established calibration method2. The effect of thapsigargin is shown as bar diagram. (B-D) Cells were treated with control siRNA or the indicated SEC61A1-siRNA for 48 h and then transformed with either control vector (C), or SEC61A1 expression plasmid (C), or mutant SEC61A1 expression plasmid (D) as indicated. After 48 h, calcium imaging experiments were carried out as in Figs 4 and 5A. Statistical analysis of the changes in the cytosolic Ca2+ concentration after the addition of thapsigargin in experiments such as presented in A are shown. P values &lt; 0.001 were defined as significant by unpaired t test and are indicated by three asterisks (***), n.s., not significant. The numbers of cells that were analyzed are indicated. Average values are given, error bars represent standard errors of the means (s.e.m.). We note that these examples were adapted from ref. 13.
<img src="/files/ftp_upload/2730/2730fig6.jpg" alt="Figure 6"/> 
Figure 6. These data indicate that the release of nascent chains from the Sec61 complex indeed leads to calcium release from the ER and the formation of a calcium nanodomain around the cytosolic mouth of the Sec61 complex. This calcium is bound by calmodulin and calcium-calmodulin closes the Sec61 complex.&#x201D;

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	<li>Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signalling.">Calcium signalling.</a> Cell. 131, 1047-1058 (2007).</li><li>Lomax, R. B., Camello, C., Van Coppenolle, F., Petersen, O. H., Tepikin, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+and+physiological+Ca2++leak+from+the+endoplasmic+reticulum+of+pancreatic+acinar+cells.">Basal and physiological Ca2+ leak from the endoplasmic reticulum of pancreatic acinar cells.</a> J. Biol. Chem. 277, 26479-26485 (2002).</li><li>Van Coppenolle, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome-translocon+complex+mediates+calcium+leakage+from+endoplasmic+reticulum+stores.">Ribosome-translocon complex mediates calcium leakage from endoplasmic reticulum stores.</a> J. 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Biophys. 462, 115-121 (2007).</li><li>Ong, H. L., Liu, X., Sharma, A., Hegde, R. S., Ambudkar, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Ca2++release+via+the+ER+translocon+activates+store+operated+calcium+entry.">Intracellular Ca2+ release via the ER translocon activates store operated calcium entry.</a> Pflugers Arch. Eur. J. Physiol. 453, 797-808 (2007).</li><li>Amer, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocon+closure+to+Ca2++leak+in+proliferating+vascular+smooth+muscle+cells.">Translocon closure to Ca2+ leak in proliferating vascular smooth muscle cells.</a> Am. J. Physiol. Heart Circ. 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L., Hendershot, L. M., Johnson, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+mechanism+underlying+BiP-mediated+gating+of+the+Sec61+translocon+of+the+endoplasmic+reticulum.">The molecular mechanism underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum.</a> J. Cell Biol. 168, 389-399 (2005).</li><li>Aneiros, E., Philipp, S. E., Lis, A., Freichel, M., Cavalie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Ca2++signalling+by+Na+/Ca2++exchangers+in+mast+cells.">Modulation of Ca2+ signalling by Na+/Ca2+ exchangers in mast cells.</a> J. Immunol. 174, 119-130 (2005).</li><li>Gross, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPC5+is+a+Ca2+-activated+channel+functionally+coupled+to+Ca2+-selective+ion+channels.">TRPC5 is a Ca2+-activated channel functionally coupled to Ca2+-selective ion channels.</a> J. Biol. Chem. 284, 34423-34432 (2009).</li><li>Erdmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+calmodulin+with+Sec61a+limit+Ca2++leakage+from+the+endoplasmic+reticulum.">Interaction of calmodulin with Sec61a limit Ca2+ leakage from the endoplasmic reticulum.</a> EMBO J. 30, 17-31 (2011).</li></ol>

No conflicts of interest declared.

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling. Here, we have described an experimental system that allows us to directly address the role of one potential Ca2+ leak channel and to characterize its putative regulatory mechanisms13. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.
We note that only viable cells should be analyzed and that overall viability should be above 80%. Therefore, cell viability is routinely evaluated employing Nuclear-ID blue/green cell viability reagent according to the manufacturer&#x2019;s protocol. Furthermore, statistical analysis of the changes in the cytosolic Ca2+ concentration is mandatory. Therefore, the experiments have to be carried out for four different batches of cells and two coverslips with at least 20 cells have to be analyzed for each condition in a single experiment. We note that the various experiments have to be carried out at the same time after seeding on the cover slips.

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 
 </tr>
 <tr>
 	<td>DMEM+GlutaMAX</td>
 <td>Invitrogen</td>
 <td>31966</td>
 
 </tr>
 <tr>
 	<td>OptiMEM+GlutaMAX</td>
 <td>Invitrogen</td>
 <td>51985</td>
 
 </tr>
 <tr>
 	<td>FBS</td>
 <td>Biochrom</td>
 <td>S0115</td>
 
 </tr>
 <tr>
 	<td>Penicillin/ Streptomycin</td>
 <td>PAA</td>
 <td>P11-010</td>
 
 </tr>
 <tr>
 	<td>HiPerFect</td>
 <td>Qiagen</td>
 <td>301707</td>
 
 </tr>
 <tr>
 	<td>Fugene HD</td>
 <td>Roche Diagnostics</td>
 <td>04709713</td>
 
 </tr>
 <tr>
 	<td>Nuclear-ID Blue/Green cell viability reagent</td>
 <td>Enzo Life Sciences</td>
 <td>ENZ-53004</td>
 
 </tr>
 <tr>
 	<td>FURA-2 AM</td>
 <td>Invitrogen</td>
 <td>F-1221</td>
 
 </tr>
 <tr>
 	<td>Puromycin</td>
 <td>Sigma</td>
 <td>P 7255</td>
 
 </tr>
 <tr>
 	<td>Thapsigargin</td>
 <td>Invitrogen</td>
 <td>T-7459</td>
 
 </tr>
 <tr>
 	<td>Ophiobolin A</td>
 <td>Enzo Life Sciences</td>
 <td>ALX-270-109</td>
 
 </tr>
 <tr>
 	<td>Trifluoperazine</td>
 <td>Sigma</td>
 <td>T 6062</td>
 
 </tr>
 <tr>
 	<td>Countess&reg; Automated Cell Counter</td>
 <td>Invitrogen</td>
 <td>&nbsp;</td>
 
 </tr>
 <tr>
 	<td>Typhoon-Trio imaging system</td>
 <td>GE Healthcare</td>
 <td>&nbsp;</td>
 
 </tr>
 <tr>
 	<td>TE2000-S microscope
with DS-5Mc camera
</td>
 <td>Nikon</td>
 <td>&nbsp;</td>
 
 </tr>
 <tr>
 	<td>iMIC microscope 
with polychrome V
</td>
 <td>Till Photonics</td>
 <td>&nbsp;</td>
 
 </tr>
 
 </tbody>
</table>

live cell calcium imaging combined with sirna mediated gene silencing identifies ca2+ leak channels in the er membrane and their regulatory mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Watch this Scientific Journal Video about Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms at JoVE

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

live-cell-calcium-imaging-combined-with-sirna-mediated-gene-silencing

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18.0K 조회수.  Saarland University. endoplasmic reticulum은 단백질 biogenesis과 칼슘 항상성에 중요한 역할을합니다. 우리는 우리 Ca2 + 누출 채널의 역할을 주소로하고 putative 규제 메커니즘을 특징 수있는 실험적인 체제를 구축했습니다. 이 시스템은 siRNA 중재 유전자 입을 라이브 세포 Ca2 + 이미징을 포함한다.

비디오: Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.