大鼠血清适用于哺乳动物全胚胎培养的制备
Summary
Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.
Abstract
Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.
Introduction
各种型号的动物被用于发育生物学在分子和细胞水平研究发育机制。例如，两栖类和鸟类物种已被广泛地用作传统的模型动物，都适合直接操纵的胚胎，因为这些胚胎发育母体之外。相对于这些动物，哺乳动物胚胎生长在母亲的子宫，并且在稍后阶段的增长是关键取决于子宫的功能。因此，它通常是难以直接操纵哺乳动物胚胎如那些来自小鼠，大鼠在早期阶段。在20世纪60年代，丹尼斯新成立的哺乳动物全胚胎培养（WEC）技术，使用WEC设备有连续供氧和热控制1。职业英语运动中，小鼠和大鼠胚胎能生长在体外 ，（ 即子宫外）。虽然WEC技术是通过添加各种CHEMIC常用于畸形人的化合物到培养基中，这种技术也被用在各种发育生物学研究，以了解在哺乳动物2-4独特发育机制。例如，WEC是通过使用荧光染料5，细胞移植6，并通过脂质转染7和电穿孔8-13基因导入结合其他技术，如细 胞标记，在野生型和突变体胚胎。
最近， 在子宫内的操作已经被用于分析在啮齿动物胚胎的发育过程的后期和已结合电穿孔技术14-16。然而，这些技术不适合于由于难以在早期阶段实现精确局部注射的DNA溶液成胚胎的着床后和妊娠中期胚胎的操纵。虽然超声引导细胞移植和注射病毒载体的成早期胚胎（ 即，E8.5-E-9.5中的鼠标）， 在子宫内已有报道以前17,18，优秀的技能都需要有很高的成功率进行这些实验。因此，WEC与高可达体外具有优势对于小鼠和大鼠胚胎的操控。
从雄性大鼠制备立即离心（IC）的大鼠血清通常用于WEC介质。当胚培养在植入后阶段（ 即，早于E8.0在小鼠或E10.0大鼠），合成培养基和IC大鼠血清的混合物通常被用作用于WEC 19的介质。但是，要培养胚胎在妊娠中期（ 即 。，E8.0-12.5在小鼠胚胎或E10.0-E14.5大鼠胚胎），100％的血清应作为一个媒介，因为目前没有可用的替代介质允许胚胎在体外正常生长超过2天。
高品质的大鼠血清的制备是在WEC的实验实现重复性的关键一步。相比延迟离心，集成电路具有减少溶血时的血清被收集通过挤压纤维蛋白凝块，因为大部分的红血细胞已被分离的纤维蛋白凝块的好处。如溶血性大鼠血清不能支持大鼠和小鼠胚胎的正常生长，血清的使用即时离心制备优选使用延迟离心制备。我们的协议包含其他协议相比，两个额外的步骤18-20（ 即 ，，储存在冰上收集的血液前，先离心和保持所收集的血液样本，2小时，4℃第一次离心后）。前者步骤可延缓血液凝块的形成，而后者步骤促进纤维蛋白凝块，易于压缩的固化。因此，我们的协议可以由初学者。然而，重现采血及血清提取准确地单指协议的书籍是非常困难的19-21。在这个视频文章中，我们展示我们的标准协议，以获得最佳的IC大鼠血清，其中包括来自雄性大鼠和提取血清离心的腹主动脉采血的准备。
Protocol
Representative Results
Discussion
在WEC实验可重复性的结果是依赖于使用高品质IC大鼠血清和准确的胚胎解剖技术3。不要缩短采血前禁食期间，因为禁食是必要的血液中的葡萄糖浓度标准化。雌性大鼠不应该被用来进行血清的制备，因为激素水平在雌性动物按动情周期的变化，并且这种变化在雌二醇的激素不适用于胚胎培养物。此外，极其脂肪酸雄性大鼠不应该用于血清制备，以避免在血液中大量脂肪污染。
<p class="jo…Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Mr. Hajime Ichijo for video recording and helpful advice concerning editing the video. We also thank the Osumi lab members for animal care. This work is supported by a Grant-in-Aid for Scientific Research on Innovative Area, Neural Diversity and Neocortical Organization, from MEXT of Japan (to N. O.). T. K. was supported by a Research Fellowship of JSPS for Young Scientists.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Isoflurane | Abbott | B506 | For anesthesia of rats. |
| Large scissors | Napox | B-7H | Stelized scissors for cutting the skin and muscle of rats. |
| Curved forceps | Napox | A-3-2 | Stelized forceps for picking up the skin of rats and squeezing the fibrin clot. |
| Sprague-Dawley (SD) rat | Charles Rivers Laboratories | Retired male rats from colony in the lab or purchased retired male rats. | |
| Syringe (20 ml) | TERUMO | SS-29ESZ | |
| Needle (21G x 5/8") | TERUMO | NN-2116R | |
| Sterile test (spitz) tube (10 ml) | ASIAKIZAI | 1101C000B-10 | For collection of boold |
| Sterile disposable pipette | Eiken Chemical | CD2000 No.4 | |
| Sterilie disposable tube (15 ml) | Falcon | 2196 |
References
- New, D. A. T., Copp, A. J., Cockroft, D. L. Introduction. Mammalian Postimplantation Embryos. A Practical Approach. , 1-14 (1990).
- Eto, K., Takakubo, F. Improved development of rat embryos in culture during the period of craniofacial morphogenesis. J Craniofac Genet Dev Biol. 5, 351-355 (1985).
- Takahashi, M., Osumi, N. The method of rodent whole embryo culture using the rotator-type bottle culture system. J Vis Exp. (42), e2170 (2010).
- New, D. A. Whole embryo culture, teratogenesis, and the estimation of teratologic risk. Teratology. 42, 635-642 (1990).
- Matsuo, T., et al. A mutation in the Pax-6 gene in rat small eye is associated with impaired migration of midbrain crest cells. Nat Genet. 3, 299-304 (1993).
- Nomura, T., Holmberg, J., Frisen, J., Osumi, N. Pax6-dependent boundary defines alignment of migrating olfactory cortex neurons via the repulsive activity of ephrin A5. Development. 133, 1335-1345 (2006).
- Yamamoto, M., et al. Nodal antagonists regulate formation of the anteroposterior axis of the mouse embryo. Nature. 428, 387-392 (2004).
- Inoue, T., et al. Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development. Development. 128, 561-569 (2001).
- Takahashi, M., Osumi, N. Pax6 regulates specification of ventral neurone subtypes in the hindbrain by establishing progenitor domains. Development. 129, 1327-1338 (2002).
- Calegari, F., Haubensak, W., Yang, D., Huttner, W. B., Buchholz, F. Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA. Proc Natl Acad Sci U S A. 99, 14236-14240 (2002).
- Takahashi, M., Nomura, T., Osumi, N. Transferring genes into cultured mammalian embryos by electroporation. Dev Growth Differ. 50, 485-497 (2008).
- Pryor, S. E., Massa, V., Savery, D., Greene, N. D., Copp, A. J. Convergent extension analysis in mouse whole embryo culture. Methods Mol Biol. 839, 133-146 (2012).
- Kikkawa, T., et al. Dmrta1 regulates proneural gene expression downstream of Pax6 in the mammalian telencephalon. Genes Cells. 18, 636-649 (2013).
- Tabata, H., Nakajima, K. Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex. 신경과학. 103, 865-872 (2001).
- Fukuchi-Shimogori, T., Grove, E. A. Neocortex patterning by the secreted signaling molecule FGF8. Science. 294, 1071-1074 (2001).
- Saito, T., Nakatsuji, N. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev Biol. 240, 237-246 (2001).
- Turnbull, D. H. Ultrasound backscatter microscopy of mouse embryos. Methods Mol Biol. 135, 235-243 (2000).
- Pierfelice, T. J., Gaiano, N. Ultrasound-guided microinjection into the mouse forebrain in utero at E9.5. J Vis Exp. (45), e2047 (2010).
- Quinlan, G. A., Khoo, P. L., Wong, N., Trainor, P. A., Tam, P. P. Cell grafting and labeling in postimplantation mouse embryos. Methods Mol Biol. 461, 47-70 (2008).
- Nagy, A., Gertsenstein, M., Vinterstein, K., Behringer, R., Nagy, A., Gertsenstein, M., Vinterstein, K., Behringer, R. Roller culture of postimplantation embryos. Manipulating the Mouse Embryo: A Laboratory Manual. , 237-241 (2003).
- Garcia, M. D., Udan, R. S., Hadjantonakis, A. K., Dickinson, M. E. Preparation of rat serum for culturing mouse embryos. Cold Spring Harb Protoc. 2011, 391-393 (2011).
- Brown-Woodman, P. D., Ritchie, H. E., Korabelnikoff, A., Emmanuel, C. Replacement of ether with alternate volatile anesthetics for collection of rat serum used in embryo culture. Toxicol In Vitro. 18, 719-724 (2004).
- Gray, J., Ross, M. E. Neural tube closure in mouse whole embryo culture. J Vis Exp. (56), e3132 (2011).
- Machado, C. B., et al. Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons. Development. 141, 784-794 (2014).
- Glanville-Jones, H. C., Woo, N., Arkell, R. M. Successful whole embryo culture with commercially available reagents. Int J Dev Biol. 57, 61-67 (2013).
- Ornoy, A., Yacobi, S., Yaffee, P. A simple method of culture of 11.5-day-old rat embryos in DMEM/F12 and 20% fetal bovine serum. J Anat. 203, 419-423 (2003).
- Moore-Scott, B. A., Gordon, J., Blackburn, C. C., Condie, B. G., Manley, N. R. New serum-free in vitro culture technique for midgestation mouse embryos. Genesis. 35, 164-168 (2003).
- Kalaskar, V. K., Lauderdale, J. D. Mouse Embryonic Development in a Serum-free Whole Embryo Culture System. J Vis Exp. (85), e50308 (2014).
