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研究的重要工具。用于显微注射的DNA片段通常是由启动子、编码序列和末端序列组成的微基因构建，或者是由5‘调控区和3’末端区域组成的基因组片段。更长的DNA片段包含更多的信息量，对于在空间和时间上精确的基因表达具有优势，以模拟原始的基因表达模式。因此，在小鼠基因组中引入大片段DNA的方法是研究长达几百个碱基的基因和基因家族所必需的。我们先前的研究表明，注射到受精卵中的片段通过在其末端的重叠区域进行同源重组而组合成更大的片段。在本项目中，我们通过聚合酶链式反应辅助克隆和测序，确定了同源重组和异源重组连接区的核苷酸序列。注射的非重叠片段以头到尾的方向组合，并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通过同源重组结合在一起，在连接处有少量核苷酸替换，但没有检测到核苷酸的缺失。这些结果表明，通过同源重组重组大片段是将大分子DNA引入小鼠基因组的一种有用的方案。

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.