Smith A J, Kalogerakis B
Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK.
Nucleic Acids Res. 1991 Dec;19(25):7161-70. doi: 10.1093/nar/19.25.7161.
A method is described for detecting targeted events at the mu heavy chain gene which relies on co-conversion (or co-exchange) of a point mutation with a selectable marker contained on a replacement vector. The vector, designed for application to IgM producing hybridomas, contains a single nucleotide change within the region of homology with the target gene which encodes a different allotypic determinant of IgM. In a model system where homologous recombination corrected a defective mu gene, the length of homology between this nucleotide change and the position of the double strand break in the vector was found to have a critical influence on the co-conversion frequency. In the vector design ultimately used for targeting in hybridomas, one in 1000-2000 stable transformants produced IgM with the allotype encoded by the exogenous DNA, and Southern blot analysis confirmed that these were derived by targeted integration. The sensitivity of the screening procedure using a monoclonal antibody specific to this allotype enabled a targeted clone to be detected in a pool of stable transformants when present at a frequency at least as low as one per cent. Several different modifications of the target locus were obtained as a consequence of alternative crossover positions and, in some cases, vector DNA concatenation.
本文描述了一种在重链基因处检测靶向事件的方法，该方法依赖于点突变与置换载体上所含选择标记的共转化（或共交换）。该载体设计用于应用于产生IgM的杂交瘤，在与靶基因的同源区域内包含单个核苷酸变化，该变化编码IgM的不同同种异型决定簇。在同源重组纠正缺陷重链基因的模型系统中，发现该核苷酸变化与载体中双链断裂位置之间的同源长度对共转化频率有关键影响。在最终用于杂交瘤靶向的载体设计中，每1000 - 2000个稳定转化体中有一个产生具有外源DNA编码同种异型的IgM，Southern印迹分析证实这些是通过靶向整合产生的。使用针对该同种异型的单克隆抗体的筛选程序的灵敏度使得当靶向克隆以至少百分之一的频率存在于稳定转化体库中时能够被检测到。由于替代的交叉位置以及在某些情况下载体DNA串联，获得了靶位点几种不同的修饰。
