Rohan R M, King D, Frels W I
US Department of Agriculture, Beltsville Agricultural Research Center, MD 20705.
Nucleic Acids Res. 1990 Oct 25;18(20):6089-95. doi: 10.1093/nar/18.20.6089.
When microinjected foreign genes integrate into the genomes of mice, multiple copies are frequently found clustered together at one location. How they concatamerize--by the integration of large linearized concatamers that are formed by simple end-to-end linkage, by circularization of individual DNA fragments and recombination, or by some other means--is not understood. In the transgenic animals studied thus far by ourselves and others, integration frequency and transgene copy number do not seem to be significantly influenced by the complementarity of the ends of the DNA fragments that have been microinjected. We have utilized PCR amplification and DNA sequence analysis to study selected transgene junctions at the nucleotide level. In two transgenic mice carrying the synthetic RSVcat gene (injected with noncomplementary overhangs on the fragment ends), ends were 'nibbled' from 1 to 62 bases before being joined to an adjacent gene copy. Repeated dinucleotides, providing the most minimal of homologies, are present in half of the characterized junctions. Determination of the relative copy number of the junctions in each mouse supports the idea that transgene complexes can undergo additional rearrangements after the initial formation event.
当显微注射的外源基因整合到小鼠基因组中时，经常会发现多个拷贝聚集在一个位置。它们是如何连接成串的——是通过简单的端对端连接形成的大型线性化连接体的整合、单个DNA片段的环化和重组，还是通过其他方式——目前尚不清楚。在我们自己和其他人迄今为止研究的转基因动物中，显微注射的DNA片段末端的互补性似乎对整合频率和转基因拷贝数没有显著影响。我们利用聚合酶链反应（PCR）扩增和DNA序列分析在核苷酸水平上研究了选定的转基因连接点。在两只携带合成RSVcat基因的转基因小鼠中（片段末端注射了非互补的突出端），末端在与相邻基因拷贝连接之前被“蚕食”了1至62个碱基。在一半已鉴定的连接点中存在重复的二核苷酸，提供了最少量的同源性。确定每只小鼠中连接点的相对拷贝数支持了这样一种观点，即转基因复合体在初始形成事件后可以经历额外的重排。
