The basic studies on the insertional mutation in transgenic mice

为了确定DNA片段在注入受精卵原核后如何整合到宿主基因组中，我们分析了两个或三个共享重叠区域的片段共注射产生的转基因小鼠的重组情况。由pUC19质粒BamHI位点的人乳头瘤病毒16型DNA组成的环状质粒pSy14 (10.6 kb)，用3对限制性内切酶酶切，每个片段在5′和3′端具有重叠区域，用琼脂糖凝胶电泳纯化。将三个片段以等摩尔量混合在一起，共同注射到受精卵中，产生转基因小鼠。尾部DNA的Southern分析和质粒拯救实验表明，共注入片段在重叠区域通过同源重组重组为原pSy14质粒序列，然后将序列重复序列整合到宿主染色体中。两个片段共注射在转基因小鼠中也产生同源重组事件。在大肠杆菌中进行的质粒拯救实验表明，由于基因组序列通常在CG基序的许多C残基中甲基化，甲基化依赖限制系统（Methylation Dependent Restriction System）的突变需要将质粒DNA从转基因基因组有效地恢复到能态细胞中。质粒拯救的效率在分离基因组DNA的组织中是不同的。

To determine how DNA fragments integrate into the host genome after microinjection into the pronuclei of fertilized mouse eggs, we analyzed the recombination status in transgenic mice produced by coinjection with two or three fragments which shared overlap-ping regions each other. Circular plasmid DNA, pSy14 (10.6 kb), which is composed of human papillomavirus type 16 DNA in BamHI site of pUC19 plasmid, was digested with 3 pairs of restriction enzymes and each fragment which possessed overlapping regions in its 5' and 3' ends was purified by agarose gel electrophoresis. Three fragments were mixed together with equal molar amounts and coinjected into fertilized mouse eggs to generate transgenic mice. Southern analysis and plasmid rescue experiments of tail DNA showed that the coinjected fragments were reconstructed to original pSy14 plasmid sequences by homologous recombination in their overlapping regions and then the tandem repeats of result-ant pSy14 integrated into host chromosome. Coinjection with two fragments also produced homologous recombination events in trans-genic mice. Plasmid rescue experiments into Escherichia coli indicated that mutations in the Methylation Dependent Restriction System were required for the efficient recovery of plasmid DNA from transgenic genome into competent cells because genomic sequences were usually methylated in many C residues of the CG motif. Efficiency of plasmid rescue was varied among the tissues from which genomic DNA were isolated.