Sprangers Anthony J, Freeman Brian T, Kouris Nicholas A, Ogle Brenda M
Department of Biomedical Engineering, University of Wisconsin-Madison, USA.
J Vis Exp. 2012 Jan 4(59):e3581. doi: 10.3791/3581.
The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts(1-4). More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation(5-7). Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease. Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo(8-15). We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system(16, 17), we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo. To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.
在整个进化过程中，后生动物利用两种或更多相同类型细胞的融合能力形成了许多复杂器官，包括骨骼肌、骨骼和胎盘。当代研究表明，相同类型细胞的融合赋予了增强的功能。例如，胎盘的滋养层细胞融合形成合体滋养层时，合体滋养层比未融合的滋养层细胞更能有效地跨母婴屏障运输营养物质和激素（1-4）。最近的研究表明，不同类型细胞的融合可以引导细胞命运。细胞命运通过融合发生的“逆转”或改变曾被认为仅限于细胞培养系统。但是干细胞移植的出现使我们和其他人发现，干细胞可以在体内与体细胞融合，并且融合促进干细胞分化（5-7）。因此，细胞融合是一个受调控的过程，能够促进细胞存活和分化，因此可能对发育、组织修复甚至疾病发病机制至关重要。限制细胞融合研究的是缺乏适当的技术来：1）准确识别融合产物；2）随着时间推移追踪融合产物。在这里，我们提出了一种新方法，通过融合时诱导生物发光来解决这两个限制（图1）；生物发光在体内可以高灵敏度检测到（8-15）。我们利用一种构建体，其编码萤火虫荧光素酶（Photinus pyralis）基因，该基因位于由LoxP序列侧翼的终止密码子旁边。当表达该基因的细胞与表达Cre重组酶蛋白的细胞融合时，LoxP位点被切割，终止信号被切除，从而允许荧光素酶转录。因为信号是可诱导的，假阳性信号的发生率非常低。与利用Cre/LoxP系统的现有方法不同（16, 17）我们引入了一个“活体”检测信号，从而首次有机会在体内追踪细胞融合的动力学。为了证明该方法，普遍表达Cre重组酶的小鼠作为接受者，接受转染了一种构建体的干细胞，该构建体用于在一个侧翼有LoxP序列的终止密码子下游表达荧光素酶。干细胞通过心肌内注射进行移植，移植后进行活体图像分析，以追踪心脏和周围组织中融合产物随时间的存在情况。这种方法可以适用于分析发育、疾病或成人组织修复任何阶段的任何组织类型中的细胞融合。
