小鼠胚胎生殖细胞的分离和推导
Summary
胚胎生殖细胞分化成原始生殖细胞在早期发展阶段的能力，是一个完美的模型，以解决我们的假设，对癌症和不育症。该协议显示了如何从发展中国家性腺的原始生殖细胞中分离出来后10.5-11.5天coitum小鼠胚胎。
Abstract
胚胎生殖细胞的能力（例如），以区分在早期发育阶段，是一个完美的模型，以解决我们的假设，对癌症和不育症的原始生殖细胞（PGCS），后来进入配子。该协议显示了如何在10.5-11.5天coitum（DPC）的小鼠胚胎来自发展中国家性腺的原始生殖细胞分离。发展中国家从小鼠胚胎性腺脊（C57BL6J）分离机械破碎与胶原酶，然后镀上了一个小鼠胚胎成纤维细胞饲养层（MEF – CF1）以前分裂与丝裂霉素C灭活淘汰赛媒体的存在，并辅以白血病抑制，因子（LIF），碱性成纤维细胞生长因子（bFGF），干细胞因子（SCF）。使用这些优化方法盈科鉴定，隔离，并建立培养条件允许超过40天，EG细胞的长期培养。胚胎生发细胞株显示胚胎的表型多能状态的常用标记和表达。文化的生发细胞的分离和衍生提供了一个工具，以了解体外他们的发展提供了机会，在遗传和表观遗传一级监察累积损伤后接触氧化应激。
Protocol
第1部分：孕鼠剖腹使用颈椎脱位，安乐死在10.5-11.5 DPC怀孕C57BL6J雌性小鼠。 抗菌肥皂与清洁腹部，然后，刮胡子。 剃须后，用生理盐水溶液冲洗腹部。 然后，用无菌纱布擦干腹部。 盖鼠标与一个新的无菌领域。 制作腹切口，使用镊子和解剖剪刀。 识别和清除腹腔整个子宫。小鼠胚胎将子宫内可见。 转移到培养皿中弥漫着的D – PBS的子宫，?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者想承认了宝贵的帮助：尼尔第一手稿的编辑和Kourtney威尔金森博士;露西信通博士，博士Brigit Willeford和迈克Basett在密歇根州立大学ALAC的认可鼠标设施，培训和动物服务的援助;德韦恩明智博士为他的援助显微镜和图像采集;塞萨尔蒙罗伊，汉娜Swoope，协助他们在密歇根州立大学与视频生产和Bobbie赫德尔斯顿。这项研究是由研究机构和密西西比州立大学生物科学系办公室。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| 10.5 – 11.5 dpc pregnant C57BL6J female mouse crossed with the same background male mouse. | ||||
| Mouse Fibroblast MEF-CF1 ATCC | SCRC-1040 | |||
| Dulbecco’s Phosphate Buffered Saline (D-PBS) | Invitrogen | 14190/086 | ||
| Collagenase /Dispase Solution | Roche | 269638 | ||
| Gelatin | Sigma | G1890 | ||
| Mitomycin C | Sigma | M4287 | ||
| Trypsin EDTA | Invitrogen | 25200-072 | ||
| MEF Medium: Dulbecco Medium Eagle Modified (D-MEM) GLUTAMAX High glucose | Invitrogen | 10566024 | ||
| 10% Fetal Bovine Serum | Invitrogen | 16000/044 | ||
| 1% of antibiotic-antimycotic | Invitrogen | 15420/096 | ||
| Knockout Media: 80% knockout D-MEM | Invitrogen | 10829/018 | ||
| 20% Knockout serum Invitrogen Cat. No. 1028/028, 200mM L-glutamine Invitrogen | 25130/081 | with β-mercaptoethanol Sigma Catalogue Number: M7522 | ||
| 1X non essential amino acid solution | Invitrogen | 11140/050 | ||
| 1% antibiotic-antimycotic | Invitrogen | 15420/096 | supplemented with grow factor as 2500 U of Leukemia inhibitory factor (mLIF-ESGRO) Millipore Catalogue Number: ESG1106 | |
| 40 ng/ml of Recombinant Murine Stem Cell Factor (SCF) ReproTech | 250-03 | |||
| 20 ng/ml of Basic Fibroblast Growth factor (bFGF) | Invitrogen | 13256-029 | ||
| Corning center well culture dish 60mm | Fisher | 07-200-79 | 1.5 ml eppendorf tube, and 15 ml falcon tubes |
Forceps No. 14 and 15, scalpel 35 mm, dissection scissors, surgical fields, antimicrobial soap, saline solution 0.9 %, gauze, ice foam container, razor blade, scalpel 35 mm, fine forceps No. 4 and 5, fine teasing needle with handle, filter paper sheets, and pulled glass pipettes.
Equipment: dissecting stereomicroscope, light source Schott Fostec, inverted microscope, micropipette 10 and 200ul, centrifuge, bio safety Cabinet.
Equipment: dissecting stereomicroscope, light source Schott Fostec, inverted microscope, micropipette 10 and 200ul, centrifuge, bio safety Cabinet.
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Tags
IsolationDerivationMouse Embryonic Germinal CellsPrimordial Germinal CellsDifferentiationCancerInfertilityProtocolDeveloping GonadsMouse EmbryosDissociationMechanical DisruptionCollagenaseMouse Embryo Fibroblast Feeder LayerMitotically InactivatedKnockout MediaLeukemia Inhibitor Factor (LIF)Basic Fibroblast Growth Factor (bFGF)Stem Cell Factor (SCF)PCG IdentificationEstablishment Of Culture ConditionsLong Term CulturesEmbryonic PhenotypePluripotent StateGenetic DamageEpigenetic DamageOxidative Stress
Citar este artigo
Moreno-Ortiz, H., Esteban-Perez, C., Badran, W., Kent-First, M. Isolation and Derivation of Mouse Embryonic Germinal Cells. J. Vis. Exp. (32), e1635, doi:10.3791/1635 (2009).