Caporale L H, Chartrain N, Tocci M, DeHaven P
Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Gene. 1990 Mar 15;87(2):285-9. doi: 10.1016/0378-1119(90)90314-h.
Protoplast fusion is a method for directly transferring cloned DNA from bacteria to mammalian cells at high efficiency. Here, we have used membrane-bound alkaline phosphatase as a reporter enzyme in a miniprotoplast fusion assay. This work demonstrates the principle that large numbers of protoplast fusions can be done simultaneously and successfully, to assay for an activity encoded by an expression vector. The technique described here circumvents key hurdles to expression cloning. This method does not require a highly sensitive assay or a way of separating a rare expressing cell from the mixture of transfected cells containing other transfected genes. With a strong promoter, the protein encoded by the undiluted transfected cDNA should be produced at at least as high a level as it is endogenously produced in the cell from which its activity was first detected. Reference clones are stored, avoiding the need to separate out the cells that are successfully transfected; this also avoids the need to repurify the DNA from the transfected cell. Because of the use of microtiter plates, it is likely that such a method could be partially automated for many types of assays.
原生质体融合是一种将克隆的DNA从细菌高效直接转移至哺乳动物细胞的方法。在此，我们在微型原生质体融合试验中使用膜结合碱性磷酸酶作为报告酶。这项工作证明了这样一个原理，即可以同时成功地进行大量原生质体融合，以检测由表达载体编码的活性。本文所述技术规避了表达克隆的关键障碍。该方法不需要高度灵敏的检测方法，也不需要从含有其他转染基因的转染细胞混合物中分离出罕见的表达细胞。有了强启动子，未稀释的转染cDNA编码的蛋白质产生水平应至少与首次检测到其活性的细胞内源性产生水平一样高。保存参考克隆，无需分离成功转染的细胞；这也避免了从转染细胞中重新纯化DNA的需要。由于使用了微量滴定板，这种方法很可能可以针对多种类型的检测进行部分自动化操作。
