Basic studies on the introduction of huge DNA molecules into fertilized mouse eggs by homologous recombination between injected fragments.

转基因小鼠的生产现在是生物医学研究和实验动物科学调查的重要工具。用于显微注射的DNA片段通常是由启动子、编码区和末端序列组成的小基因构建体，或由5'调控区和3'末端区所包含的基因组片段。更长的DNA片段包含更多的信息，这将有利于基因在空间和时间上的精确表达，以模拟原始基因的表达模式。因此，需要将巨大的DNA片段引入小鼠基因组的方法来研究数百个碱基长的基因和基因家族。我们以前的研究表明，注射到受精卵中的片段在其末端的重叠区域通过同源重组结合成更大的片段。本研究通过PCR辅助克隆和测序，确定了同源重组和异源重组结合区的核苷酸序列。将注射的非重叠片段以首尾方向组合，并且在连接处观察到几个核苷酸的缺失和替换。重叠片段在其重叠区域通过同源重组结合，并且在接合处周围观察到少量核苷酸的替换，但是，未检测到核苷酸的缺失。这些结果表明，通过同源重组的大片段的重建是一个有用的协议，为巨大的DNA分子在小鼠基因组中的引入。

Production of transgenic mice is now an important tool for the investigation of biomedical research and laboratory animal science. DNA fragments for microinjection are usually used as the minigene construct composed of promoter, coding and terminal sequences, or the genomic fragment encompassed by 5' regulatory and 3' terminal regions. Longer DNA fragment including more informations must have advantages for the precise gene expression in spatial and temporal manner to mimic the original gene expression patterns. Therefore, the method to introduce huge DNA fragments into mouse genome is required for investigation of genes and gene families which are extended for several hundred kilobase long. Our previous research indicated that the fragments injected into fertilized mouse eggs combined to constitute larger fragments by homologous recombination in the overlapping regions of their terminals. In this project we determined the nucleotide sequences in the conjunct regions of homologous and heterogenous recombination by PCR-aid cloning and sequencing. Non-overlapping fragments injected were combined in head to tail orientation, and the deletion and replacement of several nucleotides were observed in the junctions. Overlapping fragments were combined by homologous recombination in their overlapping regions and the replacement of few nucleotide was observed around the junction, however, the deletion of nucleotides was not detected. These results indicated that the reconstitution of large fragments by homologous recombination was a useful protocol for introduction of huge DNA molecules on the mouse genome.