갓 고립 된 인간의 산모 Cytotrophoblasts에 siRNA를 형질 및 EMSA 분석
Summary
이 프로토콜은 효과적으로 격리 갓 인간 융모 cytotrophoblasts에 siRNA를 형질 감염 microporation을 사용하여 이들 세포의 DNA 단백질 복합체를 식별하는 방법을 설명한다. 형질 감염된 세포는 웨스턴 블롯에 의해 모니터링 될 수 있고, EMSA는 4 일간의 배양 기간 동안 분석한다.
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
인간의 태반 기능과 개발에 관한 연구는 매우 다양한 태반 세포 집단의 분리를 최적화하기위한 프로토콜이 향상되었습니다. 최고의 연구 된 태반 세포 집단 중 하나는 매우 효율적이고 신뢰할 수있는 격리를 허용 최적화 된 프로토콜에서 도움이되고 연구되는의 융모 cytotrophoblasts을, 남아있다. 이것은 또한 이러한 형질 전환 및 프로모터 연구 등의 실험의 수를 허용했다. 이전에 설명 된 프로?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
이 작품은 캐나다의 국립 과학 및 공학 연구위원회 (NSERC) (# 298527) (BB)에서 교부금에 의해 지원되었다. CT는 기관 FARE 장학금에 의해 지원되었다. AV는 NSERC 그레이엄 벨 박사에 의해 지원되었다 장학금. BB는 인간 Retrovirology에서 캐나다 연구 의장에게 (2 단계)을 개최했다. 텍스트를 수정에 도움을 베아트릭스 Beisner 감사합니다.
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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