Nickoloff J A, Spirio L N, Reynolds R J
Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque 87131, USA.
Mol Biotechnol. 1998 Oct;10(2):93-101. doi: 10.1007/BF02760857.
Plasmid-based transfection assays provide a rapid means to measure homologous and nonhomologous recombination in mammalian cells. Often it is of interest to examine the stimulation of recombination by DNA damage induced by radiation, genotoxic chemicals, or nucleases. Transfection is frequently performed by using calcium phosphate coprecipitation (CPP), because this method is well suited for handling large sample sets, and it does not require expensive reagents or equipment. Alternative transfection methods include lipofection, microinjection, and electroporation. Since DNA strand breaks are known to stimulate both homologous and nonhomologous recombination, the induction of nonspecific damage during transfection would increase background recombination levels and thereby reduce the sensitivity of assays designed to detect the stimulation of recombination by experimentally induced DNA damage. In this article, we compare the stimulatory effects of nuclease-induced double-strand breaks (DSBs) on homologous and nonhomologous recombination for molecules transfected by CPP and by electroporation. Although electroporation yielded fewer transfectants, both nonhomologous and homologous recombination were stimulated by nuclease-induced DSBs to a greater degree than with CPP. Ionizing radiation is an effective agent for inducing DNA strand breaks, but previous studies using CPP generally showed little or no stimulation of homologous recombination among plasmids damaged with ionizing radiation. By contrast, we found clear dose-dependent enhancement of recombination with irradiated plasmids transfected using electroporation. Thus, electroporation provides a higher signal-to-noise ratio for transfection-based studies of damage-induced recombination, possibly reflecting less nonspecific damage to plasmid DNA during transfection of mammalian cells.
基于质粒的转染分析提供了一种快速方法来测量哺乳动物细胞中的同源和非同源重组。通常，研究辐射、基因毒性化学物质或核酸酶诱导的DNA损伤对重组的刺激作用是很有意义的。转染通常采用磷酸钙共沉淀法（CPP）进行，因为这种方法非常适合处理大量样本，并且不需要昂贵的试剂或设备。其他转染方法包括脂质体转染、显微注射和电穿孔。由于已知DNA链断裂会刺激同源和非同源重组，转染过程中诱导的非特异性损伤会增加背景重组水平，从而降低旨在检测实验诱导的DNA损伤对重组刺激作用的分析的灵敏度。在本文中，我们比较了核酸酶诱导的双链断裂（DSB）对通过CPP和电穿孔转染的分子的同源和非同源重组的刺激作用。尽管电穿孔产生的转染子较少，但核酸酶诱导的DSB对非同源和同源重组的刺激程度均高于CPP。电离辐射是诱导DNA链断裂的有效因子，但先前使用CPP的研究通常表明，用电离辐射损伤的质粒之间同源重组的刺激作用很小或没有。相比之下，我们发现用电穿孔转染的受辐照质粒的重组有明显的剂量依赖性增强。因此，电穿孔为基于转染的损伤诱导重组研究提供了更高的信噪比，这可能反映了在哺乳动物细胞转染过程中质粒DNA受到的非特异性损伤较少。
