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

この作品は、カナダの国立科学と工学研究評議会（NSERC）（＃298527）（BB）からの助成金によってサポートされていました。 CTは、制度FAREの奨学金によってサポートされていました。 AVはNSERC・グラハム・ベル博士によってサポートされていました奨学金。 BBは、ヒトRetrovirology（ティア2）にカナダの研究の椅子を開催しました。テキストの改訂で助けをベアトリクスBeisnerに感謝します。

UQAM倫理委員会は、センターHospitalier Universitaireモントリオール（モントリオール、カナダ）のサンリュック病院の倫理委員会のガイドラインに従っているこれらのプロトコルを、承認しました。参加者は、インフォームドコンセントフォームに署名しました。 1.ミディアム調製と原発絨毛細胞栄養層の分離<ol><li> 25mMのHEPES、10％ウシ胎児血清（FBS）および1％ペニシリン/ストレプ?...

<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>

ヒト胎盤機能と開発に関する研究が大幅に様々な胎盤細胞集団の分離を最適化することを目的としたプロトコルによって改善されました。最もよく研​​究さ胎盤細胞集団の一つは研究が大幅に効率的で信頼性の高い分離を可能にする最適化されたプロトコルから恩恵を受けている、絨毛細胞栄養芽層のまま。これはさらに、そのようなトランスフェクションおよびプロモーターの研究など?...

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>&#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 height="22" style="height:22px;">Personal Molecular Imager (PMI) System</td>
			<td>BioRad</td>
			<td></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.

満期妊娠からの新鮮な胎盤は、プロトコルのセクションで説明した実験のセットを行うために、ヒト初代絨毛細胞栄養層を分離するために使用されました。それらの単離に続いて、我々は最初のサイトケラチン7マーカー（ 図1）の使用を介して細胞栄養層の純度を分析しました。細胞調製物は、このように、モノクローナル抗サイトケラチン7抗体を用い?...

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

このプロトコルは、効率的に新たに単離されたヒト絨毛細胞栄養芽層でのsiRNAのトランスフェクションマイクロポを使用して、これらの細胞におけるDNA-タンパク質複合体を同定するための方法が記載されています。トランスフェクトされた細胞は、ウェスタンブロットによりモニターすることができ、EMSAは、4日間の培養期間中に分析します。

新たに単離したヒト絨毛細胞栄養芽層に対するsiRNAトランスフェクションおよび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 ...

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Single Molecule Analysis of Laser Localized Psoralen Adducts

レーザーローカライズソラレン付加物の単一分子解析

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

マイクロ Rna の齧歯動物の神経筋疾患を治療する遺伝子組換えアデノ随伴ウイルス血清型 9 を使用しての全身配信

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

点突然変異修復 CRISPR/Cas9 とひと細胞での SsODN によって触媒の機能として敷地内変異原性を調べるための標準方法論

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

キイロタマホコリカビの選択に基づいて細胞と細菌の成長の遺伝子工学

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

音響ナノディスペンス技術を用いたハイスループットDNAプラスミド多重化とトランスフェクション

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

非正規の開いたリーディング ・ フレームから翻訳された蛋白質を発表する OpenProt データベースを利用した質量分析を用いたプロテオミクス解析

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

マウス横紋筋肉腫から単離された原発性腫瘍細胞からの腫瘍球誘導と治療

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

ゲノムワイド往復半角化分析によるサッカロマイセス酵母の種間の熱耐性の遺伝学的マッピング

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 ...