Potter H, Weir L, Leder P
Proc Natl Acad Sci U S A. 1984 Nov;81(22):7161-5. doi: 10.1073/pnas.81.22.7161.
We have developed a general method for introducing cloned genes into mammalian cells that affords substantial benefits over current technology. It is simple, rapid, and applicable to many (perhaps all) cell types, including those that are refractory to traditional transfection procedures. The method involves exposure of a suspension of cells and cloned DNA to a high-voltage electric discharge. In a model application of this transfection procedure, we have studied the expression of cloned human and mouse Ig kappa genes stably introduced into mouse pre-B cells and fibroblasts. We find that there is a B-cell-specific enhancer-activator region in the J-C intron of the human kappa gene that is necessary for efficient transcription of the cloned gene in mouse pre-B lymphocytes. This suggests that both the DNA element and the proteins required for its regulatory activity have been highly conserved in evolution and that these elements operate at the pre-B-cell stage of immunocyte development, a stage that precedes productive kappa gene rearrangement.
我们已经开发出一种将克隆基因导入哺乳动物细胞的通用方法，该方法相比现有技术具有显著优势。它操作简单、速度快，适用于许多（或许是所有）细胞类型，包括那些对传统转染程序具有抗性的细胞类型。该方法是将细胞悬液和克隆的DNA暴露于高压放电环境中。在这个转染程序的一个模型应用中，我们研究了稳定导入小鼠前B细胞和成纤维细胞中的克隆人源和鼠源Ig κ基因的表达情况。我们发现，人κ基因的J-C内含子中存在一个B细胞特异性增强子激活区域，该区域对于克隆基因在小鼠前B淋巴细胞中的高效转录是必需的。这表明，DNA元件及其调控活性所需的蛋白质在进化过程中高度保守，并且这些元件在免疫细胞发育的前B细胞阶段发挥作用，这一阶段先于κ基因的有效重排。
