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

Dette arbeidet ble støttet av et stipend fra National Sciences og Engineering Research Council of Canada (NSERC) (# 298527) (BB). CT ble støttet av en institusjonell FARE stipend. AV ble støttet av en NSERC Graham Bell Ph.D. stipend. BB holdt en Canada Research Chair i Human Retrovirology (Tier 2). Takk til Beatrix Beisner om hjelp i å revidere teksten.

Den UQAM etiske komité har godkjent disse protokollene, som er i samsvar med retningslinjene for etisk komité for St-Luc Hospital av Centre Hospitalier Universitaire de Montréal (Montréal, Canada). Deltakerne undertegnet et informert samtykkeskjema. 1. Medium Fremstilling og isolering av Primære villøse Cytotrophoblasts <ol><li> Fremstille dyrkningsmedium for humane primære villous cytotrophoblasts ved å supplere Dulbeccos modifiserte Eagles medium (DMEM) med 25 mM HEPES, 10% føtalt bovint seru...

<ol>
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Studier av menneskelig morkakefunksjon og utvikling har blitt kraftig forbedret av protokoller som tar sikte på å optimalisere isolering av ulike placental celle populasjoner. En av de beste studert morkakecellepopulasjon forblir de villous cytotrophoblasts, studiet av noe som har stor nytte av optimaliserte protokoller som tillater effektiv og pålitelig isolasjon. Dette har ytterligere tillot en rekke eksperimenter, så som transfeksjon og promotor studier. Ved hjelp av en tidligere beskrevet protokoll 3,...

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>Montr&#233;al Biotech</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.

Ferske placentas fra sikt svangerskap ble brukt til å isolere humane primære villous cytotrophoblasts å gjennomføre settet med eksperimenter som presenteres i protokollen delen. Etter deres isolasjon, vi først analysert renheten av cytotrophoblasts gjennom bruk av den cytokeratin-7 markør (figur 1). Cellepreparater ble så farget ved å bruke et monoklonalt anti-cytokeratin-7 antistoff. Figur 1 viser resultatene fra et typisk forsøk etter rensing ...

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

Denne protokollen beskriver en fremgangsmåte for effektiv transfeksjon av siRNA i nylig isolerte humane villous cytotrophoblasts ved hjelp microporation og identifisering av DNA-proteinkomplekser i disse cellene. Transfekterte celler kan overvåkes ved hjelp av Western blot-analyser og EMSA i løpet av 4 dagers kultur tid.

siRNA Transfeksjon og EMSA Analyser på nylig isolerte humane villøse Cytotrophoblasts

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

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

Genteknologi Dictyostelium discoideum cellene basert på valget og vekst 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

Høy-gjennomstrømning DNA plasmider multipleksing og Transfeksjoner bruke akustisk Nanodispensing teknologi

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

Masse massespektrometri-baserte Proteomikk analyser bruker OpenProt databasen å avsløre romanen proteiner oversatt fra ikke-kanoniske åpent lesing rammer

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 avledning og behandling fra primære tumor celler isolert fra 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 kartlegging av Thermotolerance forskjeller mellom arter av Saccharomyces gjær via Genova-bredt gjensidige Hemizygosity Analysis

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 ...