小鼠胚胎肾的解剖与培养
Summary
该方案描述了一种从小鼠胚胎中分离和培养元肾小球的方法。
Abstract
该方案的目的是描述一种用于解剖，分离和培养小鼠睾丸的初步方法。
在哺乳动物肾脏发育期间，两个祖细胞组织，输尿管芽和间质间质介导并相互诱导细胞机制，最终形成肾脏的收集系统和肾单位。由于哺乳动物胚胎生长在宫内，因此观察者无法接近，已经开发出器官培养。通过这种方法，可以研究肾脏器官发生期间的上皮 – 间质相互作用和细胞行为。此外，可以研究先天性肾脏和泌尿生殖道畸形的起源。经仔细解剖后，将met ric r are are are are onto onto that that on。。。。。。。。。。。。。。。。。。。。。。。但是，必须意识到条件是人造并可能影响组织中的新陈代谢。此外，由于外植体中存在的细胞外基质和基底膜，测试物质的渗透可能受到限制。
器官文化的一个主要优点是实验者可以直接进入器官。该技术便宜，简单，并且可以进行大量的修改，例如添加生物活性物质，遗传变异体的研究以及先进成像技术的应用。
Introduction
The mammalian kidney is derived from two primordial structures with mesodermal origin: the tubular epithelial ureteric bud and the metanephric mesenchyme. During nephrogenesis, the ureteric bud invades the metanephric mesenchyme and branches to form the collecting system. The metanephric mesenchyme gives rise to the epithelial elements of the nephrons. These processes occur in a precisely timed and spatially coordinated manner and are initiated by reciprocal inductive mechanisms. Both tissue components communicate and affect the other’s cell morphogenesis.
In the 1920s, it was Boyden who performed the in vivo obstruction of the mesonephric duct in chicken, providing the first indication of inductive interactions as separated nephric blastema fail to differentiate1. At about the same time, the first successful attempts to culture chicken nephric rudiments in a hanging drop were published. Subsequently, the organ culture was developed to study tissue interactions in mammalian organogenesis. In the 1950s, Grobstein developed a technique in which metanephric rudiments could be cultured on a filter. This technique was modified by Saxén, who placed the filter on a Trowell-type screen in a culture dish1. Over the years, many modifications and applications for organ culture have emerged. The method described here is based on Saxén’s technique but is simplified, as the filters float free on the medium and the diameter of the culture well only slightly exceeds the diameter of the filter, limiting unwanted movement of the filter.
Whole-organ culture is a classical, cheap, and simple but powerful tool to investigate cellular processes and intercellular communication during organogenesis. Organ culture allows for treatment with biological agents, such as growth factors, antibodies, antisense oligonucleotides, viruses, and peptides, as well as with pharmaceutical compounds and other chemicals. Also, gene function may be studied using explants derived from genetically modified mice or using inducible gene inactivation technology, such as the Cre-loxP system. This allows for the study of genetic mutations that cause embryonic lethality prior to the development of the kidney. Organ culture can also be combined with fluorescent tagging for gene function or lineage tracing and modern imaging techniques, which enable real-time monitoring of cell behavior2.
In the specific example provided here, the effect of EphrinB2-activated Eph-receptor signaling on the branching morphology of the ureteric bud was investigated. The morphology of the EphA4/EphB2 double-knockout mice suggested several severe defects in kidney development, which were detectable as early as embryonic day 11 (E11) and involved the ureteric bud, the ureter, and the common nephric duct3. Signaling via Eph receptors requires the clustering of the ligand-receptor dimer4. To over-activate Eph signaling, the kidney rudiments from E11.5 mouse embryos were cultured in the presence of clustered recombinant EphrinB2-Fc. EphrinB2 is a known ligand for the EphA4 receptor, which is expressed in the ureteric bud tips3.
Protocol
Representative Results
Discussion
该手稿描述了一种从小鼠胚胎中分离出发育中的甲基化成核细胞并培养器官基因的方法。这种方法是由Grobstein 8和Saxén9,10开发的一种标准技术，并被许多其他11,12修改和修改。该方法的成功主要取决于解剖的持续时间，随着剥离时间的延长，外植体存活和诱导电位降低。在清洁周围组织的肾脏基础时，还要注意不要损伤间质。间质间质的损伤通常是外植体生长?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
作者感谢Leif Oxburgh和Derek Adams慷慨分享他们的知识，Leif Oxburgh对手稿有帮助的评论，StefanWölfl和UlrikeMüller的技术支持和Saskia Schmitteckert，Julia Gobbert，Sascha Weyer和Viola Mayer在实验室。这项工作得到了“生物学家公司” （CP）发展部门的支持。
Materials
| DMEM/F-12 | Thermo Fisher Scientific | 21331020 | |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140148 | |
| GlutaMAX Supplement | Thermo Fisher Scientific | 35050061 | |
| DPBS, calcium, magnesium | Thermo Fisher Scientific | 14040117 | use for dissection |
| holo-Transferrin human | Sigma-Aldrich | T0665 | |
| Insulin-Transferrin-Selenium (ITS -G) (100X) | Thermo Fisher Scientific | 41400045 | |
| Paraformaldehyde | Sigma-Aldrich | 158127 | |
| Amphotericin B solution | Sigma-Aldrich | A2942 | |
| Triton X-100 | Sigma-Aldrich | X100 | |
| Sodium azide | Sigma-Aldrich | S8032 | |
| Thimerosal | Sigma-Aldrich | T5125 | |
| Propyl gallate | Sigma-Aldrich | 2370 | |
| Mowiol 4-88 | Sigma-Aldrich | 81381 | |
| Glycerol | Sigma-Aldrich | G5516 | |
| Biotinylated Dolichorus Biflorus Agglutinin | Vector Laboratories | B-1035 | |
| Alexa488 conjugated Streptavidin | Jackson Immuno Research | 016-540-084 | |
| Recombinant Mouse Ephrin-B2 Fc Chimera Protein, CF | R&D Systems | 496-EB | |
| Recombinant Human IgG1 Fc, CF | R&D Systems | 110-HG-100 | |
| Goat Anti-Human IgG Fc Antibody | R&D Systems | G-102-C | |
| Phosphate buffered saline tablets | Sigma-Aldrich | P4417 | use for fixation and immunostaining |
| Dumont #5, biologie tips, INOX, 11cm |
agnthos.se | 0208-5-PS | 2 pairs of forceps are needed |
| Iris scissors, straight, 12cm | agnthos.se | 03-320-120 | |
| Dressing Forceps, straight, delicate, 13cm |
agnthos.se | 08-032-130 | |
| Petri dishes Nunclo Delta treated | Thermo Fisher Scientific | 150679 | |
| TMTP01300 Isopore Membrane Filter, polycarbonate, Hydrophilic, 5.0 µm, 13 mm, white, plain | MerckMillipore | TMTP01300 | |
| Nunclon Multidishes 4 wells, flat bottom |
Sigma-Aldrich | D6789-1CS | |
| Microscope cover glass24x50mm thickn. No.1.5H 0.17+/-0.005mm | nordicbiolabs | 107222 | |
| Cover glasses No.1.5, 18x18mm | nordicbiolabs | 102032 | |
| Slides ~76x26x1, 1/2-w. ground plain | nordicbiolabs | 1030418 | |
| VWR Razor Blades | VWR | 55411-055 | |
| 50 mL centrifuge tubes | Sigma-Aldrich | CLS430828 | |
| 15 mL centrifuge tubes | Sigma-Aldrich | CLS430055 | |
| Whatman prepleated qualitative filter paper, Grade 113V, creped | Sigma-Aldrich | WHA1213125 | |
| Fixed stage research mircoscope | Olympus | BX61WI | |
| Black 6 inbred mice, male, C57BL/6NTac | Taconic | B6-M | |
| Black 6 inbred mice,female, C57BL/6NTac | Taconic | B6-F | |
| Greenough Stereo Microscope | Leica | Leica S6 E |
Referencias
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