阿的Cre - LOX P为检测细胞融合重组方法在体内</em
Summary
一个在生活随着时间的推移的生物体细胞融合的方法来跟踪描述。该方法利用CRE -<em> LoxP位</em>重组荧光素酶表达诱导细胞融合后。产生荧光信号可以检测到使用生物光子成像系统的检测〜1000细胞周围组织的敏感性，在生活的有机体。
Abstract
已使用两个或两个以上的相同类型的细胞融合的能力，在整个进化的后生动物，形成了许多复杂的器官，包括骨骼肌肉，骨骼和胎盘。现代研究表明，同一类型的细胞融合赋予了增强的功能。例如，当胎盘保险丝的滋养层细胞形成的合体，合体能够更好地运输营养和激素跨比 1-4非稠合滋养的母胎屏障。最近的研究表明，不同类型的细胞融合可以直接细胞的命运。曾经被认为“回归”或修改融合细胞的命运是有限的细胞培养系统。但干细胞移植的到来我们发现，干细胞可以在体内与体细胞融合，融合促进干细胞分化 5-7 。因此，细胞融合是一个监管过程中Capable促进细胞的存活和分化，从而可以发展，修复组织和疾病的发病机制的极端重要性。
限制细胞融合的研究，是缺乏适当的技术：1）准确地识别融合的产品，以及2）随着时间的推移跟踪融合产品。这里我们提出一种新的方法来解决双方的局限性，通过融合后诱导生物发光（图1）;可生物发光检测灵敏度高，在 体内 8-15，我们利用一个构造编码萤火虫荧光素酶基因（Photinus pyralis）放在相邻一个终止密码子两侧LoxP位序列的细胞与细胞表达Cre重组酶蛋白基因的保险丝时，LoxP位网站裂解，使荧光素酶转录停止信号切除，由于信号是induciBLE，假阳性信号的发病率是很低的。与现有方法利用CRE / LoxP系统16，17不同的是，我们已成立了一个“活”的探测信号，从而承受的首次机会跟踪体内细胞融合的动力学。
为了证明这种方法，无处不在表达Cre重组酶的小鼠干细胞转染与构造来表达一个floxed终止密码子的荧光素酶的下游接受者。干细胞移植通过内注射和移植活体图像分析后进行跟踪存在随着时间的推移的心脏和周围组织的融合产品。这种方法可以适应在任何组织类型分析，在任何阶段的发展，疾病或成人组织修复细胞融合。
Protocol
Discussion
这里的方法允许的话，第一次，在生物识别离散细胞融合和时空分析，包括小动物。后续的生物光子图像分析的方法相结合的Cre – LoxP位重组。跟踪不仅是细胞与细胞融合的方法是适合，而且病毒的细胞融合等可以跟踪病毒感染的证明。图像分析是快速，有可能图像的多个小动物的同时，融合检测融合在其相应的微环境，特定的细胞伙伴的频率和LoxP位停止LoxP位的萤光素酶质粒转染效?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者想感谢慷慨提供H1的干细胞，并添哈克博士，博士Gouqing宋和吉尔科克女士威斯康星大学心血管生理学博士Peiman Hemmati（医学系，威斯康星大学麦迪逊分校）执行鼠标手术的核心设施。这项工作是由国家科学基金会通过一个研究生研究奖学金布莱恩弗里曼和美国国立卫生研究院R21 HL089679的支持。
Materials
| Name of reagent | Company | Catalogue number | Comments |
| Neon Transfection System | Invitrogen, Carlsbad, CA | MPK5000 | |
| Neon 100 μL Kit | Invitrogen, Carlsbad, CA | MPK10025 | Contains R and E Buffer |
| a-MEM powder | Invitrogen, Carlsbad, CA | 12000-022 | |
| Fetal Bovine Serum (FBS) | Hyclone, Logan UT | SH30070.03 | |
| B6.C-Tg(CMV-cre)1Cgn/J | Jackson Laboratory, Bar Harbor, ME | 006054 | |
| Trypsin 10X | Fisher Scientific, Forest Lawn, NJ | MT-25-054-Cl | |
| L-Glutamine | Fisher Scientific, Forest Lawn, NJ | 25030-081 | |
| D-Luciferin | Caliper Life Sciences, Hopkinton, MA | 122796 | |
| Xenogen Biophotonic Imaging System | Caliper Life Sciences, Hopkinton, MA | IVIS Spectrum | |
| Sodium Biocarbonate | Sigma Aldrich, St. Louis, MO | S6014-500G | |
| Non-essential Amino Acids | Invitrogen, Carlsbad, CA | 11140-050 |
Riferimenti
- Bernirschke, K. K. P. . Pathology of the Human Placenta. , (2000).
- Hoshina, M., Boothby, M., Boime, I. Cytological localization of chorionic gonadotropin alpha and placental lactogen mRNAs during development of the human placenta. J. Cell. Biol. 93, 190-198 (1982).
- Johansen, M., Redman, C. W., Wilkins, T., Sargent, I. L. Trophoblast deportation in human pregnancy–its relevance for pre-eclampsia. Placenta. 20, 531-539 (1999).
- Redman, C. W., Sargent, I. L. Placental debris, oxidative stress and pre-eclampsia. Placenta. 21, 597-602 (2000).
- Ogle, B. M. Spontaneous fusion of cells between species yields transdifferentiation and retroviral transfer in vivo. FASEB. J. 18, 548-550 (2004).
- Nygren, J. M. Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation. Nat. Med. 10, 494-501 (2004).
- Nygren, J. M. Myeloid and lymphoid contribution to non-haematopoietic lineages through irradiation-induced heterotypic cell fusion. Nat. Cell. Biol. 10, 584-592 (2008).
- Kutschka, I. Adenoviral human BCL-2 transgene expression attenuates early donor cell death after cardiomyoblast transplantation into ischemic rat hearts. Circulation. 114, I174-I180 (2006).
- Min, J. J. In vivo bioluminescence imaging of cord blood derived mesenchymal stem cell transplantation into rat myocardium. Ann. Nucl. Med. 20, 165-170 (2006).
- Malstrom, S. E., Tornavaca, O., Meseguer, A., Purchio, A. F., West, D. B. The characterization and hormonal regulation of kidney androgen-regulated protein (Kap)-luciferase transgenic mice. Toxicol. Sci. 79, 266-277 (2004).
- Weir, L. R. Biophotonic imaging in HO-1.luc transgenic mice: real-time demonstration of gender-specific chloroform induced renal toxicity. Mutat. Res. 574, 67-75 (2005).
- Rajashekara, G., Glover, D. A., Banai, M., O’Callaghan, D., Splitter, G. A. Attenuated bioluminescent Brucella melitensis mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) confer protection in mice. Infect. Immun. 74, 2925-2936 (1090).
- Kadurugamuwa, J. L. Noninvasive biophotonic imaging for monitoring of catheter-associated urinary tract infections and therapy in mice. Infect. Immun. 73, 3878-3887 (2005).
- Ryan, P. L., Youngblood, R. C., Harvill, J., Willard, S. T. Photonic monitoring in real time of vascular endothelial growth factor receptor 2 gene expression under relaxin-induced conditions in a novel murine wound model. Ann. N.Y. Acad. Sci. 1041, 398-414 (2005).
- Zhu, L. Non-invasive imaging of GFAP expression after neuronal damage in mice. Neurosci. Lett. 367, 210-212 (2004).
- Noiseux, N. Mesenchymal stem cells overexpressing Akt dramatically repair infarcted myocardium and improve cardiac function despite infrequent cellular fusion or differentiation. Mol. Ther. 14, 840-850 (2006).
- Ajiki, T. Composite tissue transplantation in rats: fusion of donor muscle to the recipient site. Transplant Proc. 37, 208-209 (2005).
- Trivedi, P., Hematti, P. Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells. Exp. Hematol. 36, 350-359 (2008).
- Ogle, B. M., Cascalho, M., Platt, J. L. Biological implications of cell fusion. Nat. Rev. Mol. Cell. Biol. 6, 567-575 (2005).
- Collins, T. J. ImageJ for microscopy. BioTechniques. 43, 25-30 (2007).
