This work was supported by a grant from the National Sciences and Engineering Research Council of Canada (NSERC) (#298527) (BB). CT was supported by an institutional FARE scholarship. AV was supported by a NSERC Graham Bell Ph.D. scholarship. BB held a Canada Research Chair in Human Retrovirology (Tier 2). Thanks to Beatrix Beisner for help in revising the text.

The UQAM ethics committee has approved these protocols, which are in accordance with the guidelines of the ethics committee of St-Luc Hospital of the Centre Hospitalier Universitaire de Montr&#233;al (Montr&#233;al, Canada). Participants signed an informed consent form.
1. Medium Preparation and Isolation of Primary Villous Cytotrophoblasts
<ol>
	<li>Prepare culture medium for human primary villous cytotrophoblasts by supplementing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) with 25 mM HEPES, 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin. Filter the prepared medium through sterile 0.22 &#181;m filter and store in a -20 &#176;C freezer in aliquots of 50 ml.</li>
	<li>Isolate primary villous cytotrophoblasts from fresh placentas following standard protocols 3.
	<ol>
		<li>Using pliers, remove the fetal membrane from the tissue by gently scratching it off, while being careful not to remove villous tissue. Cut the remaining placental villi in cubes of about 3 x 3 cm using scalpel and scissors. Thoroughly wash with water containing 0.9% NaCl to remove maternal blood.
		<ol>
			<li>Holding each cube with pliers, carefully remove the villous tissue from remaining vessels and fibrous tissue using scissors.</li>
			<li>For 15 to 30 mg of villous tissue, digest four times in Hank&#39;s balanced salt solution (HBSS) containing trypsin (from 9.6 x 105 to 1.8 x 106 U; final concentration 1.2 mg/ml) and deoxyribonuclease I (DNase I) (final concentration 200 &#181;g/ml) for 30 min at 37 &#176;C in a water bath with continuous shaking. 
			Note: Use 150 ml total volume for the first digestion, 100 ml for the second digestion and 75 ml for the two remaining digestions.</li>
		</ol>
		</li>
		<li>Collect the supernatant containing dispersed cells and centrifuge at 1,250 x g for 15 min without brake. Remove the supernatant and the resuspend cells in 1 ml of prewarmed DMEM containing 1% penicillin/streptomycin.</li>
		<li>Layer the dispersed cells on top of a discontinuous 5% - 70% polyvinylpyrrolidone-coated silica gradient (see Table 1 for gradient preparation) and centrifuge for 23 min at 507 x g without brake. Remove the density layer between 1.017 to 1.033 by aspiration and collect the layers between 40% and 50% in the gradient with a new pipette. Wash cells extensively with 10 ml of prewarmed DMEM containing 1% penicillin/streptomycin. 
		Note: The collected fraction corresponds to densities between 1.048 and 1.062, which harbors trophoblasts.</li>
		<li>Count the cells using a hemocytometer. Briefly, mix cells and add 10 &#181;l in a new microcentrifuge tube. Add 10 &#181;l of trypan blue and mix gently again. Draw up the cell suspension and fill the hemocytometer chambers. Count the cells under 10X objective.</li>
		<li>Place 1 x 106 and 4.5 x 106 cells into separate tubes for use in sections 1.3 and 2, respectively. Seed the remaining cells at a density of 1.5 x 106 cells/well in supplemented DMEM medium and culture at 37 &#176;C and 5% CO2 for a maximum of 4 days for other experiments.</li>
	</ol>
	</li>
	<li>Evaluation of the purity of isolated primary villous cytotrophoblasts
	<ol>
		<li>Spin 1 x 106 of the freshly isolated cytotrophoblasts in microcentrifuge tubes at 300 x g. Discard the supernatant and add 1 ml of prewarmed phosphate-buffered saline (PBS). Centrifuge cells at 300 x g and remove PBS by aspiration.</li>
		<li>Add 1 ml of cold methanol to cells and incubate at -20 &#176;C for 20 min. Centrifuge cells at 300 x g for 5 min and remove methanol by aspiration. 
		Caution: Use canister mask, safety goggles and rubber gloves while handling methanol.</li>
		<li>Add 1 ml of PBS at room temperature to rehydrate cells, centrifuge at 300 x g and remove PBS by aspiration.</li>
		<li>Incubate cells in PBS containing FBS (1:50 [v/v] dilution) and a FcR blocking reagent (1:10) for 45 min at room temperature to eliminate nonspecific binding.</li>
		<li>Wash twice with 500 &#181;l of room temperature PBS, centrifuge cells at 300 x g for 5 min and remove PBS by aspiration. Resuspend cells in 100 &#181;l room temperature PBS.</li>
		<li>Incubate cells with a mouse monoclonal fluorescein isothiocyanate (FITC)-conjugated anti-human cytokeratin-7 antibody clone LP5K (1:200) or a control isotype-matched non-specific antibody in PBS containing 0.2% BSA for 45 min at room temperature in the dark. Wash cells in PBS.</li>
		<li>Analyze the cells by flow cytometry 3. Use cell preparations, which demonstrate a minimum of 96% CK-7-positive cells by flow cytometry for further experiments.</li>
	</ol>
	</li>
</ol>
2. siRNA Transfection of Human Primary Villous Cytotrophoblasts
<ol>
	<li>Transfection of human primary villous cytotrophoblasts using a microporation device
	<ol>
		<li>Prepare 6-well plates containing 2 ml of supplemented DMEM medium for the incubation of microporated cells.</li>
		<li>After isolation of primary cytotrophoblasts (see section 1.2), take an aliquot of cells in suspension and determine cell number using a hemocytometer.</li>
		<li>Transfer 1.5 x 106 cells to a microcentrifuge tube and pellet cells by centrifugation at 300 x g for 5 min at room temperature. Wash the cells with 1 ml Dulbecco&#39;s PBS (DPBS) (Mg2+, Ca2+-free) and pellet the cells by centrifugation at 300 x g for 5 min at room temperature.</li>
		<li>Remove the supernatant and resuspend the cells gently in 100 &#181;l of resuspension buffer. 
		Note: Avoid keeping cell suspension for longer than 15-30 min at room temperature, to maximize cell viability and transfection efficiency.</li>
		<li>Add 300 ng of Syncytin-2 siRNA (or control-scrambled siRNA) to the 1.5 ml microcentrifuge tube and aspirate the cell-siRNA mixture using a 100 &#181;l transfection pipette. Insert the pipette into the station (see List of Materials) and subject the cells to a 1,300 V single pulse of 30 msec.</li>
		<li>Immediately transfer the cells to 6-well plates (prepared in step 2.1.1) containing 37 &#176;C pre-warmed supplemented medium to avoid cell damage.</li>
		<li>Repeat steps 2.1.5 and 2.1.6 for the remaining samples.</li>
		<li>Gently rock the plate for 30 sec to ensure even distribution of the cells. Incubate the plate at 37 &#176;C in a humidified CO2 incubator for 24 to 48 hr. Refresh culture medium 24 hr after transfection.</li>
		<li>Evaluate transfection efficiency by Western blot analysis (see steps 3 to 6).</li>
	</ol>
	</li>
	<li>Lipofection of siRNA in human primary cytotrophoblasts
	<ol>
		<li>After isolation of villous cytotrophoblasts, seed the cells at a density of 1.5 x 106 cells per well in 6-well plates in 2 ml of culture medium and incubate for 18 hr.</li>
		<li>Dilute 300 ng of Syncytin-2-specific siRNA (or control-scrambled siRNA) and 5 &#181;l of lipid-based transfection reagent in separate 1.5 ml tubes in a total volume of 10 &#181;l of antibiotic- and serum-free medium. Incubate for 5 min.</li>
		<li>Mix the diluted reagents and incubate for an additional 20 min at room temperature to form the transfection complex. Add 90 &#181;l of antibiotic- and serum-free medium to the mix to a final volume of 100 &#181;l.</li>
		<li>Remove culture medium from the 6-well plates (step 2.2.1). Add 2 ml of fresh medium and the 100 &#181;l transfection mix to each well. Incubate the cells at 37 &#176;C in a CO2 incubator for 24 to 48 hr. Refresh culture medium 24 hr after transfection.</li>
		<li>Evaluate transfection efficiency by Western blot analysis (see steps 3 to 6).</li>
	</ol>
	</li>
</ol>
3. Preparation of Lysates from Whole Cell Culture
<ol>
	<li>Remove the media and rinse each well (from steps 2.1.8 and 2.2.4) using pre-warmed DPBS (Mg2+, Ca2+ free). Aspirate the DPBS and add 500 &#181;l of 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA). Incubate for 1-3 min at 37 &#176;C in a CO2 incubator. Stop trypsin treatment by adding 500 &#181;l of serum containing growth media. 
	Note: Use appropriate media volume according to the type of flask/dish: 1 ml per 107 cells/100 mm dish/150 cm2 flask; 0.5 ml per 5 x 106 cells /60 mm dish /75 cm2 flask.</li>
	<li>Transfer cells to a 1.5 ml microcentrifuge tube, pellet cells by centrifugation at 300 x g for 2 min at room temperature. Wash the cells with DPBS (Mg2+, Ca2+-free) and pellet cells by centrifugation at 300 x g for 2 min at room temperature.</li>
	<li>Carefully remove supernatant without disturbing the pellet. Place the tube on ice. Resuspend the pellet in 50-200 &#181;l ice-cold Radio-Immunoprecipitation Assay (RIPA) buffer (see Table 2 for buffer composition) containing freshly added protease and phosphatase inhibitors at a 1x final concentration. Briefly, vortex the tube and leave on ice for 30 min.</li>
	<li>Spin at 16,000 x g for 20 min at 4 &#176;C. Carefully place the tube on ice. Using a pipette, transfer the supernatant to a fresh pre-cooled microcentrifuge tube kept on ice. Discard the pellet.</li>
</ol>
4. Preparation of Nuclear Extracts from Cultured Cells
<ol>
	<li>For each well (step 1.2.5), wash cultured cytotrophoblasts twice with 1 ml of pre-warmed DPBS (Mg2+, Ca2+ free). Aspirate DPBS and add 500 &#181;l of 0.25% trypsin/EDTA. Incubate for 1-3 min at 37 &#176;C in a CO2 incubator and stop trypsin treatment by adding 500 &#181;l of serum containing growth media.</li>
	<li>Transfer cells to a 1.5 ml microcentrifuge tube, pellet cells by centrifugation at 300 x g for 2 min at room temperature. Wash the cells with DPBS (Mg2+, Ca2+-free) and pellet cells by centrifugation at 300 x g for 2 min at room temperature. Carefully remove all supernatant without disrupting the pellet and place the tube on ice.</li>
	<li>Extract nuclear protein according to manufacturer&#39;s instructions. Determine protein concentration of each sample using protein quantification assay (Bradford or bicinchoninic acid assay (BCA)).</li>
</ol>
5. Sample Preparation and Electrophoresis
<ol>
	<li>Determine the protein concentration for each cell extract using a protein quantification assay (for example, Bradford 8 or BCA 9).</li>
	<li>As per manufacturer&#39;s instructions (if antibodies could be used in reduction and denaturation conditions), add an equal volume of 2x Laemmli Sample Buffer (Table 2) to 20-30 &#181;g of total protein.</li>
	<li>Boil cell lysates at 100 &#176;C for 5 min. Aliquot 50-100 &#181;l of lysate to avoid repeated freeze/thaw cycles and store the remaining tubes at -20 &#176;C for future use.</li>
	<li>While the samples are heating, prepare 1 L of 1x running buffer (Table 2).</li>
	<li>Spin the samples at 16,000 x g for a few seconds and place them on ice.</li>
	<li>Load equal amount of protein into each well of a 12% SDS-PAGE gel (see Table 2 for gel composition), along with a molecular weight marker. Run the gel for 1 to 2 hr at 100 V.</li>
</ol>
6. Transferring the Protein from the Gel to the Membrane
<ol>
	<li>Activate polyvinylidene fluoride (PVDF) membrane with methanol for 1 min and rinse with 1x transfer buffer/10% methanol solution. According to manufacturer&#39;s instructions, transfer for 30 min to 1 hr depending on the protein size. 
	Note: Transfer efficiency can be assessed using Ponceau Red staining before the blocking step.</li>
</ol>
7. Antibody Staining
<ol>
	<li>Block the membrane for 1 hr at room temperature or overnight at 4 &#176;C using 5% blocking solution.</li>
	<li>Incubate the membrane with anti-Syncytin-2 antibodies (4 &#181;g/ml; 1:5,000) 6 or with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 0.4 &#181;g/ml; 1:500) in 5% blocking solution overnight at 4 &#176;C (anti-Syncytin-2) or for 45 min at room temperature. Wash the membrane three times in Tris-buffered saline Tween (TBST; see Table 2 for buffer composition) for 5 min.</li>
	<li>Incubate the membrane with appropriate horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse antibody (1:10,000) in 5% blocking buffer in TBST at room temperature for 1 hr. Wash the membrane three times in TBST for 5 min and then rinse in TBS. Detect signals using the chemiluminescence-based blotting substrate.</li>
</ol>
8. Radiolabeling of Single Stranded Oligonucleotide
<ol>
	<li>Phosphorylation reaction
	<ol>
		<li>In a microcentrifuge tube, add 50 ng of a forward oligonucleotide along with 1x T4 polynucleotide kinase buffer, 1 U T4 polynucleotide kinase and 20 &#181;Ci (&#947;-32P) ATP and make up the reaction volume to 20 &#181;l with nuclease-free water.</li>
		<li>Incubate at 37 &#176;C for 30 min. Stop the reaction by adding 5 &#181;l of 0.5 M EDTA. Make up the volume to 50 &#181;l with nuclease-free water.</li>
	</ol>
	</li>
	<li>Removal of unincorporated nucleotides from oligonucleotides
	<ol>
		<li>Prepare a G-25 column equilibrated with TE buffer (Table 2) following manufacturer&#39;s instructions. Place the column in a fresh DNase-free 1.5 ml microcentrifuge tube.</li>
		<li>Slowly add 50 &#181;l of probe (from step 8.1.2) over the resin, being careful not to disturb the resin bed. Spin for 2 min at 350 x g to collect the purified sample in the 1.5 ml microcentrifuge tube. Wash the G-25 column with 36 &#181;l of nuclease-free water and spin for 2 min at 350 x g to collect it in the microcentrifuge tube.</li>
	</ol>
	</li>
	<li>Hybridization with the complementary strand oligonucleotide
	<ol>
		<li>Transfer the radiolabeled probe (86 &#181;l) to a 1.5 ml microcentrifuge tube and add 200 ng of the reverse oligonucleotide and 1x of annealing buffer (Table 2). Heat 5 min at 95 &#176;C and leave overnight at room temperature for cooling.</li>
		<li>Prepare non-radioactive double-stranded oligonucleotides by adding 50 ng of unlabeled forward and reverse oligonucleotides and 1x of annealing buffer in a 1.5 ml microcentrifuge tube. Make up the volume to 100 &#181;l with nuclease-free water. Heat and keep at room temperature as described in step 8.3.1.</li>
	</ol>
	</li>
</ol>
9. Preparation of a Non-denaturing Polyacrylamide Gel for EMSA
<ol>
	<li>Prepare a 4% native gel (10 x 12 cm) with a 1.5 mm space-comb (see Table 2 for gel composition). Clean all glass plates using distilled water. 
	Note: It is critical that the plates be completely free of ionic detergent, like SDS.</li>
	<li>Immediately pour the acrylamide solution in between the plates and allow polymerization to proceed (approximately 30 min).</li>
	<li>Assemble the electrophoresis apparatus and fill the tank with 0.5x TBE. Pre-run the gel for 30 min to 1 hr at 150 V before sample loading.</li>
</ol>
10. DNA Binding Reaction
<ol>
	<li>In a 1.5 ml microcentrifuge tube, add 10 &#181;g of nuclear extract (step 4.3) in 1x Gel Shift Binding buffer (Table 2) and make up the final volume to 10 &#181;l with nuclease-free water. As a control, prepare a tube with no nuclear extract. For the competition reaction, add 1 &#181;l of cold non-specific or specific double-stranded oligonucleotide (step 8.3.2).</li>
	<li>Add 1 &#181;l of radioactive probe from step 8.3.1 to each reaction. Incubate the reaction at room temperature for 20 min. Add 1 &#181;l of 10x gel loading buffer per reaction and load each sample on the gel prepared in section 9.</li>
	<li>Run the gel for 1 1/2 to 2 hr at 150 V. After migration, wrap the gel and the glass in a plastic wrap and position in an exposure cassette below a phosphor screen. Leave cassette at 4 &#176;C for 24 hr.</li>
	<li>Remove the screen from the exposure cassette and insert in the phosphor imaging device for scanning.</li>
</ol>

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The authors have nothing to disclose.

Studies on human placental function and development have been greatly improved by protocols aimed at optimizing isolation of various placental cell populations. One of the best studied placental cell population remains the villous cytotrophoblasts, the study of which has greatly benefited from optimized protocols permitting efficient and reliable isolation. This has further allowed a number of experiments, such as transfection and promoter studies. Using a previously described protocol 3, pure populations of p...

Human placental dysfunction is associated with the development of several pregnancy-associated diseases like preeclampsia and intrauterine growth restriction 1. An important cell constituent of the placenta is the trophoblasts, which can be classified as either extravillous or villous cytotrophoblasts. Upon fusion, villous cytotrophoblasts further differentiate into the syncytiotrophoblast layer, a multinuclear cell structure with an important role in feto-maternal exchange and hormone production 2. Human primary villous cytotrophoblasts and their differentiated counterparts represent important biological samples and allow researchers to study a number of placenta-related processes, such as cell fusion, in culture. Furthermore, substantial efforts are ongoing to identify markers that will facilitate appropriate management and improve preventive therapies specific to pregnancy-related diseases. Laboratories routinely isolate primary human villous cytotrophoblasts from fresh placentas, using a standard isolation procedure based on trypsin digestion of placenta villi 3. As cultured cytotrophoblasts lose their capacity to proliferate and quickly differentiate in a syncytiotrophoblast-like layer upon culture 4, very efficient transfection methods and optimized analysis approaches are needed. Previous studies have determined optimal conditions of transfection of these primary cytotrophoblasts 5. Herein, a different method of siRNA transfection, which has been previously tested in this cell type 6, is presented. In comparison to a lipofection-based approach, this microporation method improves transfection, as assessed by the extent of silencing of specific genes.
Promoter and gene expression studies also provide a better understanding of placental function. Although more difficult to use owing to the short time frame for which primary villous cytotrophoblasts can be cultured, promoter analyses using standard protocols can nonetheless be addressed, as previously published 7. Electrophoretic Mobility Shift Assay (EMSA) is one of these commonly used in vitro methods, allowing for fast and easy monitoring of DNA-protein interactions. Nuclear extracts from these primary trophoblasts were used to test a region of the Syncytin-2 promoter for specific interactions. Results revealed that bound factors could be detected at different time points of culture and in a specific and reproducible manner.
Data presented in this protocol confirm that our transfection approach and the EMSA protocol can be used for isolated primary villous cytotrophoblasts and will be of great use to study the diverse functions of villous cytotrophoblasts in normal or pathological conditions.

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			<td height="23" style="height:23px;width:432px;">HBSS without&#160; Ca2+, Mg2+&#160;</td>
			<td style="width:152px;">Sigma</td>
			<td style="width:101px;">#H2387</td>
			<td style="width:133px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS (10X)&#160;</td>
			<td>Sigma</td>
			<td>#14060-057</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">DMEM High Glucose without Hepes</td>
			<td>Gibco&#160;</td>
			<td>#12100-061</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hepes (1 M)</td>
			<td>Gibco</td>
			<td>#15630-080</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin-Neomycin (100X)</td>
			<td>Gibco&#160;</td>
			<td>#15640-055&#160;</td>
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			<td height="21" style="height:21px;">Amphotericin B&#160;</td>
			<td>Sigma</td>
			<td>#A2411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Sigma&#160;</td>
			<td>#C4901</td>
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		<tr height="22">
			<td height="22" style="height:22px;">MgSO4.7H2O&#160;</td>
			<td>Sigma&#160;</td>
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			<td>GEhealthcare</td>
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			<td>Thermo Scientific</td>
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			<td>Sigma</td>
			<td>#P9416&#160;</td>
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			<td>Cell Signaling</td>
			<td>#7074</td>
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			<td height="21" style="height:21px;">BM Chemiluminescence Western Blotting Substrate (POD)</td>
			<td>Roche Diagnostic</td>
			<td>#11500708001</td>
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			<td>Life technologies</td>
			<td>#14287-080</td>
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			<td>NEB</td>
			<td>#M0201S</td>
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			<td>Life technologies</td>
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			<td>Sigma&#160;</td>
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			<td>Millipore,</td>
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			<td height="21" style="height:21px;">&#160;FcR blocking reagent&#160;</td>
			<td>&#160;Miltenyi Biotec</td>
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			<td height="21" style="height:21px;">Flow Cytometer BD Acuri system</td>
			<td>&#160;Becton Dickinson</td>
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			<td>Digital Bio</td>
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			<td height="21" style="height:21px;">Resuspension Buffer R (Neon Transfection System 100 &#181;L Kit)</td>
			<td>&#160;Life technologies</td>
			<td>MPK10096</td>
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			<td height="21" style="height:21px;">PVDF membrane&#160;</td>
			<td>Millipore</td>
			<td>IPVH00010&#160;</td>
			<td>Activate with methanol</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Anti-human GAPDH antibody</td>
			<td>&#160;Santa Cruz Biotechnology</td>
			<td>&#160;sc-137179</td>
			<td>&#160;1:500</td>
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		<tr height="32">
			<td height="32" style="height:32px;">HorseRadish Peroxidase (HRP)-conjugated goat anti-rabbit antibody or anti-mouse antibody</td>
			<td>&#160;Cell Signalling</td>
			<td>&#160;#7074&#160;</td>
			<td>&#160;1:10,000</td>
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		<tr height="21">
			<td height="21" style="height:21px;">HorseRadish Peroxidase (HRP)-conjugated goat anti-mouse antibody</td>
			<td>&#160;Cell Signalling</td>
			<td>&#160;#7076&#160;</td>
			<td>&#160;1:10,000</td>
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		<tr height="21">
			<td height="21" style="height:21px;">&#160;NE-PER Nuclear and Cytoplasmic Extraction Reagent</td>
			<td>Thermo Scientific</td>
			<td>#78833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">G-25 column&#160;</td>
			<td>GE Healthcare</td>
			<td>#27-5325-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chemiluminsescence and fluorescence imaging device</td>
			<td>Montr&#233;al Biotech</td>
			<td>Fusion FX5</td>
			<td></td>
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			<td height="21" style="height:21px;">&#160;4 % native gel</td>
			<td>Home made</td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">PBS</td>
			<td>Home made</td>
			<td></td>
			<td>1X</td>
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		<tr height="22">
			<td height="22" style="height:22px;">Personal Molecular Imager (PMI) System</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

sirna transfection and emsa analyses on freshly isolated human villous cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

Fresh placentas from term pregnancies were used to isolate human primary villous cytotrophoblasts to conduct the set of experiments presented in the Protocol section. Following their isolation, we first analyzed the purity of cytotrophoblasts through the use of the cytokeratin-7 marker (Figure 1). Cell preparations were thus stained using a monoclonal anti-cytokeratin-7 antibody. Figure 1 represents results from a typical experiment following purification...

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siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

sirna-transfection-emsa-analyses-on-freshly-isolated-human-villous

Research

JoVE Journal

Genetics

10.5K Views.  University of Quebec in Montreal.  Dit protocol beschrijft een werkwijze voor het efficiënt siRNA transfectie van vers geïsoleerde menselijke villous cytotrofoblasten behulp microporatie en het identificeren van DNA-eiwitcomplexen in deze cellen. Getransfecteerde cellen kunnen worden gecontroleerd door Western blot en EMSA analyses gedurende de 4 dagen cultuurtijd. 

Video: siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

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 ...

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite cells. Satellite cells are characterized by the expression of the transcription factor Pax7 and their location underneath the basal lamina in the resting skeletal muscle. Upon injury, satellite cells get activated, undergo self-renewal or differentiation to either form new myofibers or to fuse with damaged ones. The functionality of satellite cells in vivo can be investigated using a cardiotoxin based injury model of skeletal muscle. To study the function of one gene during the regeneration of skeletal muscle, transgenic mouse models are mostly used. Here, we present an alternative method to transgenic mice, to investigate the gene function in satellite cells during regeneration, e.g., in cases where transgenic mice are not available. We combine the cardiotoxin mediated injury of a specific skeletal muscle with the injection of a self-delivering siRNA into the regenerating muscle which is then taken up by satellite cells among other cells. Thereby, we provide a method to analyze gene function in satellite cells during regeneration under physiological conditions without the need for transgenic mice.

We describe an in vivo method to investigate the functional loss of a specific gene in satellite cells by using a combination of cardiotoxin mediated injury of skeletal muscle and the injection of a self-delivering siRNA.

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite ...

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

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