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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Genetics

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Published: September 20th, 2016

DOI:

10.3791/53995

1Department of Biological Sciences, University of Quebec in Montreal, 2Department of Pharmacology and Therapeutics, McGill University, 3Department of Clinical Sciences, University of Montreal

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

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.

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 ....

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The UQAM ethics committee has approved these protocols, which are in accordance with the guidelines of the ethics committee of St-Luc Hospital of the Centre Hospitalier Universitaire de Montréal (Montréal, Canada). Participants signed an informed consent form.

1. Medium Preparation and Isolation of Primary Villous Cytotrophoblasts

  1. Prepare culture medium for human primary villous cytotrophoblasts by supplementing Dulbecco’s Modified Eagle’s Medium (DMEM) with 25 mM HEPES, 1.......

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Fresh placentas from term pregnancies were used to isolate human primary villous cytotrophoblasts to conduct the set of experiments presented in the Protocol section. Following their isolation, we first analyzed the purity of cytotrophoblasts through the use of the cytokeratin-7 marker (Figure 1). Cell preparations were thus stained using a monoclonal anti-cytokeratin-7 antibody. Figure 1 represents results from a typical experiment following purification.......

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Studies on human placental function and development have been greatly improved by protocols aimed at optimizing isolation of various placental cell populations. One of the best studied placental cell population remains the villous cytotrophoblasts, the study of which has greatly benefited from optimized protocols permitting efficient and reliable isolation. This has further allowed a number of experiments, such as transfection and promoter studies. Using a previously described protocol 3, pure populations of p.......

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This work was supported by a grant from the National Sciences and Engineering Research Council of Canada (NSERC) (#298527) (BB). CT was supported by an institutional FARE scholarship. AV was supported by a NSERC Graham Bell Ph.D. scholarship. BB held a Canada Research Chair in Human Retrovirology (Tier 2). Thanks to Beatrix Beisner for help in revising the text.

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Name Company Catalog Number Comments
HBSS without  Ca2+, Mg2+  Sigma #H2387
HBSS (10X)  Sigma #14060-057
DMEM High Glucose without Hepes Gibco  #12100-061
Hepes (1 M) Gibco #15630-080
Penicillin-Streptomycin-Neomycin (100X) Gibco  #15640-055 
Amphotericin B  Sigma #A2411
CaCl2 Sigma  #C4901
MgSO4.7H2 Sigma  #M
Fetal bovine serum Gibco #16170-078 
Percoll  Sigma  #P-1644  For density gradient
Syncytin-2 siRNA Ambion Life technologies #AM16708
Scrambled siRNA Qiagen # SI03650318
DNase I   Sigma-Aldrich #D5025
Trypsine, type I  Sigma  #T8003
DharmaFECT Lipotransfection  reagents  GEhealthcare # T-2001-01
Trypsin/EDTA  Life technologies #25300-062
Protease Inhibitor Cocktail Roche Diagnostic #11873580001
Pierce BCA Protein Assay Kit Thermo Scientific #23225
BSA Sigma #A7906
TWEEN 20 Sigma #P9416 
Anti-rabbit IgG, HRP-linked antibody  Cell Signaling #7074
BM Chemiluminescence Western Blotting Substrate (POD) Roche Diagnostic #11500708001
DPBS  Life technologies #14287-080
T4 Polynucleotide Kinase NEB #M0201S
ATP, [γ-32P] Perkin Elmer  #BLU002A100UC 
Acrylmide Sigma  #A9099
TEMED Life technologies #17919
Ammonium Persulfate Sigma  #A3678
Anti-human cytokeratin-7  antibody clone LP5K, FITC conjugated Millipore,  CBL194F Dilution1:200 
 FcR blocking reagent   Miltenyi Biotec  130- 059-901  Dilution 1:10
Flow Cytometer BD Acuri system  Becton Dickinson
Microporator MP-100 apparatus  Digital Bio
Resuspension Buffer R (Neon Transfection System 100 µL Kit)  Life technologies MPK10096
PVDF membrane  Millipore IPVH00010  Activate with methanol
Anti-human GAPDH antibody  Santa Cruz Biotechnology  sc-137179  1:500
HorseRadish Peroxidase (HRP)-conjugated goat anti-rabbit antibody or anti-mouse antibody  Cell Signalling  #7074   1:10,000
HorseRadish Peroxidase (HRP)-conjugated goat anti-mouse antibody  Cell Signalling  #7076   1:10,000
 NE-PER Nuclear and Cytoplasmic Extraction Reagent Thermo Scientific #78833
G-25 column  GE Healthcare #27-5325-01
Chemiluminsescence and fluorescence imaging device Montréal Biotech Fusion FX5
 4 % native gel Home made
PBS Home made 1X
Personal Molecular Imager (PMI) System BioRad

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