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

Detta arbete stöddes av ett bidrag från National vetenskaplig och teknisk forskning Council of Canada (NSERC) (# 298.527) (BB). CT stöddes av en institutionell FARE stipendium. AV stöddes av en NSERC Graham Bell Ph.D. stipendium. BB höll en Canada Research Chair i Human Retrovirology (Tier 2). Tack vare Beatrix Beisner för hjälp med att revidera texten.

Den UQAM etiska kommittén har godkänt dessa protokoll, som är i enlighet med de riktlinjer den etiska kommittén i St-Luc Hospital i Centre Hospitalier Universitaire de Montréal (Montréal, Kanada). Deltagarna undertecknat ett informerat samtycke formulär. 1. Medium framställning och isolering av primära villi cytotrofoblaster <ol><li> Förbereda odlingsmedium för humana primära villösa cytotrofoblaster genom att komplettera Dulbeccos modifierade Eagles medium (DMEM) med 25 mM HEPES, 10% fetal...

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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. 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Studier av humant placenta funktion och utveckling har förbättrats avsevärt av protokoll som syftar till att optimera isolering av olika moderkakan cellpopulationer. En av de bäst studerade placenta cellpopulationen fortfarande de villösa cytotrofoblaster, studiet av vilka har stor nytta av optimerade protokoll tillåter effektiv och pålitlig isolering. Detta har ytterligare tillåtit ett antal experiment, såsom transfektion och promotorstudier. Med hjälp av en tidigare beskrivna protokollet 3, kan re...

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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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.

Färska placentor från sikt graviditeter användes för att isolera humana primära villösa cytotrofoblaster att utföra uppsättning experiment som presenteras i protokollet sektion. Efter deras isolering, först analyserade vi renheten av cytotrofoblaster genom användningen av cytokeratin-7 markör (figur 1). Cellpreparationer har således färgades med användning av en monoklonal anti-cytokeratin-7-antikropp. Figur 1 representerar resultat från e...

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

Detta protokoll beskriver en metod för att effektivt transfektera siRNA i färsk isolerade humana villösa cytotrofoblaster genom att använda mikroporation och identifiera DNA-protein-komplex i dessa celler. Transfekterade celler kan övervakas med Western blot och EMSA-analyser under 4-dagars odling tid.

siRNA Transfektion och EMSA Analyser av nyligen isolerade mänskliga villös cytotrofoblaster

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

Genteknik Dictyostelium discoideum celler baserat på urval och tillväxt på bakterier

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

Hög genomströmning DNA plasmid Multiplexing och transfektion med akustisk Nanodispenseringsteknik

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

Mass Spectrometry-baserad proteomik analyser med hjälp av databasen OpenProt att avslöja nya proteiner översatt från icke-kanoniska öppen läsning ramar

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

Tumorsphere härledning och behandling från primära tumörceller isolerade från mus Rhabdomyosarcomas

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

Genetisk kartläggning av Termotolerans skillnader mellan arter av Saccharomyces jäst via Genomomfattande ömsesidig Hemizygosity analys

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 ...