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

وأيد هذا العمل من خلال منحة من العلوم والبحوث الهندسية المجلس الوطني لكندا (NSERC) (# 298527) (BB). وأيد CT من قبل منحة دراسية FARE المؤسسية. وأيد AV التي كتبها على درجة الدكتوراه NSERC غراهام بيل منحة دراسية. عقدت BB كرسي أبحاث كندا في Retrovirology الإنسان (المستوى 2). بفضل بياتريكس Beisner للمساعدة في إعادة النظر في النص.

وافقت لجنة الاخلاق UQAM هذه البروتوكولات، التي هي وفقا للمبادئ التوجيهية للجنة الأخلاقيات من سانت لوك مستشفى مركز HOSPITALIER الجامعي مونتريال (مونتريال، كندا). وقع المشاركون على استمارة الموافقة المسبقة. 1. إعداد المتوسطة...

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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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قد تحسنت الدراسات على وظيفة المشيمة الإنسان والتنمية إلى حد كبير من قبل بروتوكولات تهدف إلى تحسين عزل مختلف السكان الخلية المشيمة. واحدة من أفضل السكان الخلية المشيمة المدروس يبقى أرومة غاذية خلوية زغبي، ودراسة التي استفادت كثيرا من البروتوكولات الأمثل السماح عزلة...

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>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.

استخدمت مشيمة جديدة من حالات الحمل المدى لعزل أرومة غاذية خلوية زغبي الإنسان الأساسية لإجراء مجموعة من التجارب المقدمة في قسم البروتوكول. بعد عزلتهم، علينا أولا بتحليل نقاء أرومة غاذية خلوية من خلال استخدام علامة cytokeratin-7 (الشكل 1). وهكذا مل...

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

يصف هذا البروتوكول وسيلة لtransfecting بكفاءة سيرنا في أرومة غاذية خلوية زغبي الإنسان المعزولة حديثا باستخدام microporation وتحديد المجمعات الحمض النووي والبروتينات في هذه الخلايا. يمكن رصد الخلايا Transfected لطخة الغربية وEMSA يحلل خلال الوقت ثقافة لمدة 4 أيام.

سيرنا ترنسفكأيشن وEMSA تحاليل المعزولة حديثا أرومة غاذية خلوية زغبي الإنسان

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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واحدة تحليل جزيء من الليزر المترجمة السورالين 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 ...

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

منهجية تقديم ميكرورنا باستخدام النمط المصلي المسيطر المؤتلف الفيروسات المرتبطة بالغدة 9 لعلاج الأمراض العصبية العضلية في القوارض

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

منهجية موحدة لدراسة المحدثة للطفرات في الموقع كدالة لإصلاح نقطة تحور تحفزها كريسبر/Cas9 وسودن في الخلايا البشرية

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

الهندسة الوراثية ديسكويديوم ديكتيوستيليوم "الخلايا استناداً إلى" التحديد والنمو في البكتيريا

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

عالية الإنتاجية الحمض النووي بلازميد تعدد الإرسال والتحويل باستخدام تكنولوجيا نانوالاستغناء الصوتية

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

تحليلات البروتيوميات الجماعي القائم على قياس الطيف الكتلي باستخدام قاعدة البيانات أوبينبروت لكشف النقاب عن البروتينات رواية مترجمة من إطارات القراءة المفتوحة غير متعارف عليه

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

اشتقاق الأورام والعلاج من الخلايا السرطانية الأولية المعزولة من الساركوما الروماتيزمية الماوس

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

رسم الخرائط الوراثية للاختلافات التسامح الحراري بين أنواع الخميرة Saccharomyces عن طريق تحليل الهيميزيغوستي على نطاق الجينوم المتبادل

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 ...