Institute for Neurophysiology, University of Cologne, Cologne, Germany.
Biotechniques. 2010 Feb;48(2):113-20. doi: 10.2144/000113352.
We have established an in vitro Cre/loxP-based assay for monitoring cell fusion events that specifically traces the transport of cytoplasm from one cell to its fusion partner. Cells with a double fluorescence vector indicate fusion with cells expressing Cre recombinase by switching expression from red to green fluorescent protein through a Cre-mediated recombination event that simultaneously activates puromycin-acetyltransferase expression. This strategy allows for both the observation and puromycin selection of indicator cells that have undergone fusion with a Cre recombinase-expressing partner. A fusion protein of Cre with estrogen receptor (ER) can be used to control Cre recombinase activity through the tamoxifen-induced translocation of the Cre-ER fusion protein to the nucleus. Here we have established a new methodology that not only allows the monitoring of the transport of cellular contents, but also enables the purification of fused cells using puromycin.
我们建立了一种基于 Cre/loxP 的体外检测方法，用于监测细胞融合事件，该方法可特异性追踪细胞质从一个细胞到其融合伙伴的转移。带有双荧光载体的细胞通过 Cre 介导的重组事件从红色荧光蛋白切换到绿色荧光蛋白的表达，从而表明与表达 Cre 重组酶的细胞融合，该重组事件同时激活嘌呤霉素乙酰转移酶的表达。该策略允许观察和嘌呤霉素选择与表达 Cre 重组酶的伴侣融合的指示细胞。Cre 与雌激素受体 (ER) 的融合蛋白可通过他莫昔芬诱导 Cre-ER 融合蛋白向核内易位来控制 Cre 重组酶的活性。在这里，我们建立了一种新的方法，不仅可以监测细胞内容物的运输，还可以使用嘌呤霉素纯化融合细胞。
