siRNA transfektion og EMSA Analyser på frisk isoleret humant villus cytotrophoblaster
Summary
Denne protokol beskriver en fremgangsmåde til effektiv transfektion af siRNA i frisk isolerede humane villøse cytotrophoblaster hjælp mikroperforering og identificere DNA-protein-komplekser i disse celler. Transficerede celler kan monitoreres ved Western blot og EMSA-analyser under 4-dages dyrkningstid.
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
Undersøgelser af menneskelig placenta funktion og udvikling er blevet stærkt forbedret af protokoller til formål at optimere isolering af forskellige placenta cellepopulationer. En af de bedst undersøgte placenta cellepopulation forbliver de villøse cytotrophoblaster, studiet af hvilken i høj grad har nydt godt af optimerede protokoller tillader effektiv og pålidelig isolation. Dette har yderligere tilladt en række forsøg, såsom transfektion og promotor- undersøgelser. Ved hjælp af en tidligere beskrevet pro…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af en bevilling fra National Sciences og Engineering Research Council of Canada (NSERC) (# 298.527) (BB). CT blev støttet af en institutionel FARE stipendium. AV blev understøttet af en NSERC Graham Bell Ph.D. stipendium. BB holdt en Canada Research Chair i Human Retrovirology (Tier 2). Tak til Beatrix Beisner om hjælp til at revidere teksten.
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 | ||
References
- Huppertz, B. Placental origins of preeclampsia: challenging the current hypothesis. Hypertension. 51, 970-975 (2008).
- Huppertz, B. IFPA Award in Placentology Lecture: Biology of the placental syncytiotrophoblast–myths and facts. Placenta. 31, S75-S81 (2010).
- Le Bellego, F., Vaillancourt, C., Lafond, J. Isolation and culture of term human cytotrophoblast cells and in vitro methods for studying human cytotrophoblast cells’ calcium uptake. Methods Mol. Biol. 550, 73-87 (2009).
- Morrish, D. W., et al. 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. Placenta. 18, 577-585 (1997).
- Forbes, K., Desforges, M., Garside, R., Aplin, J. D., Westwood, M. Methods for siRNA-mediated reduction of mRNA and protein expression in human placental explants, isolated primary cells and cell lines. Placenta. 30, 124-129 (2009).
- Vargas, A., et al. Syncytin-2 plays an important role in the fusion of human trophoblast cells. J. Mol. Biol. 392, 301-318 (2009).
- Toufaily, C., Lokossou, A. G., Vargas, A., Rassart, E., Barbeau, B. A CRE/AP-1-Like Motif Is Essential for Induced Syncytin-2 Expression and Fusion in Human Trophoblast-Like Model. PLoS One. 10, e0121468 (2015).
- Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 (1976).
- Smith, P. K., et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85 (1985).
- Blaschitz, A., Weiss, U., Dohr, G., Desoye, G. Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening. Placenta. 21, 733-741 (2000).
- Desforges, M., et al. The SNAT4 isoform of the system A amino acid transporter is functional in human placental microvillous plasma membrane. J. Physiol. 587, 61-72 (2009).
- Felgner, P. L., et al. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 84, 7413-7417 (1987).
- Guilbert, L. J., et al. Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments. Placenta. 23, 175-183 (2002).
- Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. S. Isolation and culture of term human trophoblast cells. Methods Mol. Med. 121, 203-217 (2006).
- Ma, B., Zhang, S., Jiang, H., Zhao, B., Lv, H. Lipoplex morphologies and their influences on transfection efficiency in gene delivery. J. Control. Release. 123, 184-194 (2007).
- Freeley, M., Long, A. Advances in siRNA delivery to T-cells: potential clinical applications for inflammatory disease, cancer and infection. Biochem. J. 455, 133-147 (2013).
