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, 1...

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G., Vargas, A., Rassart, E., Barbeau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CRE/AP-1-Like+Motif+Is+Essential+for+Induced+Syncytin-2+Expression+and+Fusion+in+Human+Trophoblast-Like+Model.">A CRE/AP-1-Like Motif Is Essential for Induced Syncytin-2 Expression and Fusion in Human Trophoblast-Like Model.</a> PLoS One. 10, e0121468 (2015).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Smith, P. 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L., 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=Lipofection:+a+highly+efficient,+lipid-mediated+DNA-transfection+procedure.">Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.</a> Proc Natl Acad Sci U S A. 84, 7413-7417 (1987).</li><li>Guilbert, L. J., 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=Preparation+and+functional+characterization+of+villous+cytotrophoblasts+free+of+syncytial+fragments.">Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments.</a> Placenta. 23, 175-183 (2002).</li><li>Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. 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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>
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			<td>Gibco</td>
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			<td>#P-1644&#160;</td>
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			<td>Ambion Life technologies</td>
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			<td>Sigma&#160;</td>
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			<td>GEhealthcare</td>
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			<td>&#160;Life technologies</td>
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			<td>Thermo Scientific</td>
			<td>#23225</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>#14287-080</td>
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			<td>NEB</td>
			<td>#M0201S</td>
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			<td>&#160;Santa Cruz Biotechnology</td>
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			<td>&#160;#7074&#160;</td>
			<td>&#160;1:10,000</td>
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			<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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			<td height="21" style="height:21px;">&#160;NE-PER Nuclear and Cytoplasmic Extraction Reagent</td>
			<td>Thermo Scientific</td>
			<td>#78833</td>
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			<td height="21" style="height:21px;">G-25 column&#160;</td>
			<td>GE Healthcare</td>
			<td>#27-5325-01</td>
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			<td height="21" style="height:21px;">Chemiluminsescence and fluorescence imaging device</td>
			<td>Montr&#233;al Biotech</td>
			<td>Fusion FX5</td>
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			<td height="21" style="height:21px;">&#160;4 % native gel</td>
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			<td>BioRad</td>
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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...

Watch this Scientific Journal Video about siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts at JoVE.com

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 ...

The overall goal of this protocol is to demonstrate optimal conditions for the transfection of freshly isolated primary villous cytotrophoblasts and to present a method for the identification of DNA protein interaction at promoter sites. This method can help answer key questions in the field of placenta research, such as those related to genes involves in cytotrophoblast differentiation and the regulatory mechanisms involved in their expression. The main advantage of this technique is that they're easy to perform and very efficient.Furthermore, the time needed to conduct this experiment is short and can include a lot of different samples. Demonstrating these procedures will be Chirine Toufaily a former Phd student from my lab, as well as Yong Xiao, a research associate. Here, primary villous cytotrophoblasts are isolated from fresh placentas according to standard protocols.The first step of this protocol is to analyze the purity of the preparation by FACS analysis. First, spin one times ten to the sixth of the freshly isolated cytotrophoblasts in micro centrifuge tubes at 300 times G for five minutes. Following the centrifugation, discard the supernatant.Add one milliliter of pre-warmed PBS and repeat the centrifugation. After removing the PBS by aspiration add one milliliter of cold methanol to the cells and incubate at minus 20 degrees Celsius for 20 minutes. Next, after pelleting the cells by centrifugation, remove the methanol by aspiration.Then add one milliliter of room-temperature PBS to the pellet to rehydrate the cells. After spinning the cells and removing the PBS, add PBS containing FBS with FC receptor blocking reagent and incubate for 45 minutes at room-temperature to eliminate non-specific binding. Once the incubation time has elapsed, wash the cells twice with 500 microliters of room-temperature PBS centrifuging at 300 times G for five minutes each time.After removing the last PBS wash, resuspend the cells in 100 microliters of room-temperature PBS. Then incubate the cells with a mouse monoclonal FITC conjugated anti-human cytokeratin 7 antibody clone LP5K, or a control isotype-matched non-specific antibody for 4 minutes at room-temperature in the dark. After washing the cells in PBS, analyze florescents by flow cytometry.Use cell preparations that demonstrate a minimum of 96%CK7 positive cells by flow cytometry for further experiments. To transfect human primary villous cytotrophoblasts using a microporation device first prepare six well plates containing two milliliters of supplemented DMEM medium and place them in a 37 degree Celsius tissue culture incubator. Next, transfer aliquots of 1.5 times 10 to the 6th isolated primary cytotrophoblasts to micro centrifuge tubes.Following a centrifugation to pellet the cells wash with one milliliter of Dulbecco's PBS. After removing the PBS, and gently resuspending the washed cells in 100 microliters of resuspension buffer add 300 nanograms of Syncytin-2 siRNA or controlled scrambled siRNA. And then aspirate the cell siRNA mixture using a 100 microliter transfection pipette.Now insert the pipette into the transfection station and subject the cells to a single 1300 volt pulse of 30 milliseconds duration. Then immediately transfer the cells to the previously prepared six well plates and gently rock the plate for 30 seconds to ensure an even distribution of the cells. It's very important no to make air bubble while resuspending the cells and to transfer them immediately to the medium.Otherwise this will affect that viability of cells and the transfection efficiency. Incubate the plate at 37 degrees Celsius in a humidified carbon dioxide incubator for 24 to 48 hours. First, wash the cultured cytotrophoblasts twice with one milliliter of pre-warmed DPBS.After aspirating the DPBS add 500 microliters of 0.25%Trypsin-EDTA and incubate for one to three minutes at 37 degrees Celsius in a carbon dioxide incubator. Next, stop the Trypsin treatment by adding 500 microliters of serum containing growth medium and transfer the cells from each well into a 1.5 milliliter micro centrifuge tube. Pellet cells by centrifugation at 300 times G for two minutes at room temperature.After washing and pelleting the cells once more, carefully remove all supernatant without disrupting the pellet and place the tubes on ice until protein extraction. Prepare the phosphorylation reaction in a micro centrifuge tube by adding 15 nanograms of a forward oligonucleotide along with 1XT4 Polynucleotide kinase buffer, one unit of T4 Polynucleotide kinase, and 20 microcuries of gamma-32P labeled ATP to nuclease free water, for a final volume of 20 microliters. Incubate the reaction at 37 degrees Celsius for 30 minutes.Then, stop the reaction by adding 5 microliters of 0.5 molar EDTA. Bring the volume up to 50 microliters with nuclease free water. Then proceed to the removal of unincorporated nucleotides and the hybridization of the complimentary strand according to the instructions in the written portion of the protocol.Setup the DNA binding reaction by first adding 10 micrograms of nuclear extract 1X gel shift binding buffer to nuclease free water for a final volume of 10 microliters. For the competition reaction add one microliter of cold non-specific or specific double-stranded oligonucleotide. Next, add one microliter of the radioactive oligonucleotide probe to each reaction.Incubate the reaction at room-temperature for 20 minutes. Then add one microliter of 10X gel loading buffer per reaction. Load each sample onto a non-denaturing polyacrylamide gel and run the gel for 1.5 to 2 hours at 150 volts.After migration, wrap the gel and the glass in plastic wrap. Then position the wrapped gel in an exposure cassette and place a phosphorus screen over it. Leave the cassette at 4 degrees Celsius for 24 hours.The next day remove the screen from the exposure cassette and insert it into a phosphorimaging device for scanning. Microporation of a scrambled control or a Syncytin-2 specific siRNA in villous cytotrophoblasts is compared to a lipid based transfection approach by western blot detection of Syncytin-2 and the control protein GAPDH. Syncytin-2 protein levels were normalized in terms of band intensity with corresponding GAPDH signals.Microporation conclusively leads to a more significant knockdown of Syncytin-2 expression. This image shows an EMSA analysis of nuclear extracts villous cytotrophoblasts incubated with a probe corresponding to a promoter region of Syncytin-2 with or without an excess of specific, non-specific a cold oligonucleotide for 24 or 48 hours. The Syncytin-2 promoter drived probe showed the presence of two DNA protein complexes.Addition of cold wildtype oligonucleotide competed for the formation of the complex while no competition was observed with a cold, unrelated, oligonucleotide. While attempting this procedure, it's very important to carefully handle cytotrophoblasts after transfection and to transfer them immediately to the medium. Following the EMSA procedure, a supershift assay can be performed in order to identify transcription factor that bound to DNA.After watching this video you should have a good understanding on how to handle and carefully transfect a cytotrophoblast as well as how to perform an EMSA assay with these kind of cells.

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

Research

JoVE Journal

Genetics

10.4K 조회수.  University of Quebec in Montreal. The overall goal of this protocol is to demonstrate optimal conditions for the transfection of freshly isolated primary villous cytotrophoblasts and to present a method for the identification of DNA protein interaction at promoter sites. This method can help answer key questions in the field of placenta research, such as those related to genes involves in cytotrophoblast differentiation and the regulatory mechanisms involved in their expression. The main advantage of this technique is that th...