siRNA Transfectie en EMSA Analyses op vers geïsoleerde Human Villous cytotrofoblasten
Summary
Dit protocol beschrijft een werkwijze voor het efficiënt siRNA transfectie van vers geïsoleerde menselijke villous cytotrofoblasten behulp microporatie en het identificeren van DNA-eiwitcomplexen in deze cellen. Getransfecteerde cellen kunnen worden gecontroleerd door Western blot en EMSA analyses gedurende de 4 dagen cultuurtijd.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
Studies over menselijke placenta functie en ontwikkeling zijn sterk verbeterd door protocollen die gericht zijn op het optimaliseren van de isolatie van de verschillende placenta cel populaties. Een van de beste bestudeerde placenta cel populatie blijft villous cytotrofoblasten, de studie die heeft sterk geprofiteerd van geoptimaliseerde protocollen waardoor efficiënt en betrouwbaar isolement. Dit heeft verder liet een aantal experimenten, zoals transfectie en promoter studies. Door een eerder beschreven protocol …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dit werk werd ondersteund door een subsidie van de Nationale Raad Sciences and Engineering Research van Canada (NSERC) (# 298527) (BB). CT werd ondersteund door een institutioneel FARE beurs. AV werd ondersteund door een NSERC Graham Bell Ph.D. beurs. BB hield een Canada Research Chair in Human Retrovirology (Tier 2). Met dank aan Beatrix Beisner voor hulp in de tekst te herzien.
Materials
| 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 | |
| MgSO4.7H2O | 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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