Name: sirna transfection and emsa analyses on freshly isolated human villous cytotrophoblasts
Uploaded: 2016-09-20T08:00:00
Description: Watch this Scientific Journal Video about siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts at JoVE.com

这项工作是由加拿大国家科学与工程研究理事会（NSERC）（＃298527）（BB）的资助。 CT是由一个机构FARE奖学金支持。 AV是由NSERC·格雷厄姆·贝尔博士支持奖学金。 BB召开了加拿大研究主席在人类反转录病毒学（第2层）。由于碧翠丝Beisner在修改文本的帮助。

该UQAM伦理委员会已经批准了这些协议，这是按照大学中心医院，蒙特利尔的圣 - 吕克医院（蒙特利尔，加拿大）的伦理委员会的指导方针。与会者签署了知情同意书。 1.介质制备工艺和主代绒毛滋养细胞的分离<ol><li>通过补充的Dulbecco氏改良的Eagle氏培养基（DMEM）用25mM HEPES，10％胎牛血清（FBS）和1％青霉素/链霉素制备用于人原绒毛细胞滋养细胞的培养基。过滤通过无菌0.22微米的过滤...

<ol>
	<li>Huppertz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+origins+of+preeclampsia:+challenging+the+current+hypothesis.">Placental origins of preeclampsia: challenging the current hypothesis.</a> Hypertension. 51, 970-975 (2008).</li><li>Huppertz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFPA+Award+in+Placentology+Lecture:+Biology+of+the+placental+syncytiotrophoblast--myths+and+facts.">IFPA Award in Placentology Lecture: Biology of the placental syncytiotrophoblast--myths and facts.</a> Placenta. 31, S75-S81 (2010).</li><li>Le Bellego, F., Vaillancourt, C., Lafond, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+term+human+cytotrophoblast+cells+and+in+vitro+methods+for+studying+human+cytotrophoblast+cells'+calcium+uptake.">Isolation and culture of term human cytotrophoblast cells and in vitro methods for studying human cytotrophoblast cells' calcium uptake.</a> Methods Mol. Biol. 550, 73-87 (2009).</li><li>Morrish, D. W., 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=In+vitro+cultured+human+term+cytotrophoblast:+a+model+for+normal+primary+epithelial+cells+demonstrating+a+spontaneous+differentiation+programme+that+requires+EGF+for+extensive+development+of+syncytium.">In vitro cultured human term cytotrophoblast: a model for normal primary epithelial cells demonstrating a spontaneous differentiation programme that requires EGF for extensive development of syncytium.</a> Placenta. 18, 577-585 (1997).</li><li>Forbes, K., Desforges, M., Garside, R., Aplin, J. D., Westwood, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+siRNA-mediated+reduction+of+mRNA+and+protein+expression+in+human+placental+explants,+isolated+primary+cells+and+cell+lines.">Methods for siRNA-mediated reduction of mRNA and protein expression in human placental explants, isolated primary cells and cell lines.</a> Placenta. 30, 124-129 (2009).</li><li>Vargas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syncytin-2+plays+an+important+role+in+the+fusion+of+human+trophoblast+cells.">Syncytin-2 plays an important role in the fusion of human trophoblast cells.</a> J. Mol. Biol. 392, 301-318 (2009).</li><li>Toufaily, C., Lokossou, A. 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. K., 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=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal. Biochem. 150, 76-85 (1985).</li><li>Blaschitz, A., Weiss, U., Dohr, G., Desoye, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+reaction+patterns+in+first+trimester+placenta:+implications+for+trophoblast+isolation+and+purity+screening.">Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening.</a> Placenta. 21, 733-741 (2000).</li><li>Desforges, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SNAT4+isoform+of+the+system+A+amino+acid+transporter+is+functional+in+human+placental+microvillous+plasma+membrane.">The SNAT4 isoform of the system A amino acid transporter is functional in human placental microvillous plasma membrane.</a> J. Physiol. 587, 61-72 (2009).</li><li>Felgner, P. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+term+human+trophoblast+cells.">Isolation and culture of term human trophoblast cells.</a> Methods Mol. Med. 121, 203-217 (2006).</li><li>Ma, B., Zhang, S., Jiang, H., Zhao, B., Lv, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoplex+morphologies+and+their+influences+on+transfection+efficiency+in+gene+delivery.">Lipoplex morphologies and their influences on transfection efficiency in gene delivery.</a> J. Control. Release. 123, 184-194 (2007).</li><li>Freeley, M., Long, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+siRNA+delivery+to+T-cells:+potential+clinical+applications+for+inflammatory+disease,+cancer+and+infection.">Advances in siRNA delivery to T-cells: potential clinical applications for inflammatory disease, cancer and infection.</a> Biochem. J. 455, 133-147 (2013).</li></ol>

研究人类胎盘功能和发展已通过旨在优化各种胎盘细胞群的分离方案有了很大的提高。其中最好的研究的胎盘细胞群仍是代绒毛滋养细胞，其研究有很大的优化，从许可协议的高效，可靠的隔离受益。这进一步允许了若干实验，如转染和促进剂的研究。使用先前描述的方案3中 ，可以得到主绒毛细胞滋养细胞的纯群体。 CK-7是一种中间丝蛋白，其在滋养细胞中表达。针对此蛋白的单克隆抗?...

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.

<table border="0" cellpadding="0" cellspacing="0" width="818">
	<tbody>
		<tr height="23">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepes (1 M)</td>
			<td>Gibco</td>
			<td>#15630-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin-Neomycin (100X)</td>
			<td>Gibco&#160;</td>
			<td>#15640-055&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">MgSO4.7H2O&#160;</td>
			<td>Sigma&#160;</td>
			<td>#M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td>Gibco</td>
			<td>#16170-078&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Percoll&#160;</td>
			<td>Sigma&#160;</td>
			<td>#P-1644&#160;</td>
			<td>For density gradient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syncytin-2 siRNA</td>
			<td>Ambion Life technologies</td>
			<td>#AM16708</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scrambled siRNA</td>
			<td>Qiagen</td>
			<td># SI03650318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I&#160;</td>
			<td>&#160;Sigma-Aldrich</td>
			<td>#D5025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsine, type I&#160;</td>
			<td>Sigma&#160;</td>
			<td>#T8003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DharmaFECT Lipotransfection&#160; reagents&#160;</td>
			<td>GEhealthcare</td>
			<td># T-2001-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin/EDTA</td>
			<td>&#160;Life technologies</td>
			<td>#25300-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail</td>
			<td>Roche Diagnostic</td>
			<td>#11873580001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>#23225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma</td>
			<td>#A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TWEEN 20</td>
			<td>Sigma</td>
			<td>#P9416&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-rabbit IgG, HRP-linked antibody&#160;</td>
			<td>Cell Signaling</td>
			<td>#7074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BM Chemiluminescence Western Blotting Substrate (POD)</td>
			<td>Roche Diagnostic</td>
			<td>#11500708001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS&#160;</td>
			<td>Life technologies</td>
			<td>#14287-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 Polynucleotide Kinase</td>
			<td>NEB</td>
			<td>#M0201S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATP, [&#947;-32P]</td>
			<td>Perkin Elmer&#160;</td>
			<td colspan="2">#BLU002A100UC&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acrylmide</td>
			<td>Sigma&#160;</td>
			<td>#A9099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TEMED</td>
			<td>Life technologies</td>
			<td>#17919</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Persulfate</td>
			<td>Sigma&#160;</td>
			<td>#A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human cytokeratin-7&#160; antibody clone LP5K, FITC conjugated</td>
			<td>Millipore,</td>
			<td>&#160;CBL194F</td>
			<td>Dilution1:200&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;FcR blocking reagent&#160;</td>
			<td>&#160;Miltenyi Biotec</td>
			<td>&#160;130- 059-901</td>
			<td>&#160;Dilution 1:10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow Cytometer BD Acuri system</td>
			<td>&#160;Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microporator MP-100 apparatus&#160;</td>
			<td>Digital Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVDF membrane&#160;</td>
			<td>Millipore</td>
			<td>IPVH00010&#160;</td>
			<td>Activate with methanol</td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;4 % native gel</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Home made</td>
			<td></td>
			<td>1X</td>
		</tr>
		<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.

从足月妊娠胎盘新鲜用于分离人主要代绒毛滋养细胞进行的一组中的协议部分提供实验。继他们的孤立，我们首先分析细胞滋养细胞的纯度通过使用角蛋白7标记（ 图1）。使用单克隆抗细胞角蛋白7的抗体的细胞制备物从而染色。 图1表示从以下主要绒毛细胞滋养细胞的纯化典型的实验中，通过标准的流式细胞仪分析的结果。以下的密度梯度步骤，...

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

这个协议描述了使用微孔化有效转染的siRNA在新鲜分离的人绒毛细胞滋养细胞和鉴定在这些细胞中DNA的蛋白质复合物的方法。转染的细胞可以通过Western印迹进行监测，并在为期4天的培养时间，EMSA分析。

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

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.

Research

JoVE Journal

Genetics

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

接合交配测定在接合介入转移蛋白的序列特异性分析

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

科研

遗传学

Controllable Ion Channel Expression through Inducible Transient Transfection

通过诱导瞬时转染可控离子通道表达

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

激光局部补骨脂素加合物的单分子分析

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

重组腺相关病毒9型 MicroRNA 系统分娩治疗啮齿动物神经肌肉疾病

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

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

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

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

基于细菌选择和生长的盘状细胞基因工程

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

使用声学纳米分配技术实现高通量DNA质粒多路复用和转染

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

基于质谱的蛋白质组学分析, 利用 OpenProt 数据库揭开从非规范开放阅读框架翻译的新蛋白质

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

从小鼠横纹肌瘤分离的原发性肿瘤细胞的肿瘤球衍生和治疗

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

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

通过全基因组互惠性分析,对糖母菌菌种间热耐性差异的遗传图谱

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 ...