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首页> 外文期刊>Molecular and Cellular Biology >Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.
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Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

机译：微注射到培养的哺乳动物细胞中的DNA整合的模式：注射的质粒DNA分子之间同源重组的证据。

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We examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene. (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk- and RAT-2tk- cells to the TK+ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA. Even though we demonstrated that cultured mammalian cells contain the enzymes for ligating two DNA molecules very efficiently irrespective of the sequences or topology at their ends, we found that even linear plasmid DNA was recruited into the concatemer by homologous recombination.
机译：我们检查了显微注射到培养的哺乳动物细胞核中的DNA的命运。携带可选择基因的载体的序列组成和物理形式影响DNA介导的转化效率。在猿猴病毒40的复制起点附近或在禽肉瘤原病毒的长末端重复序列中引入序列，将其引入含有单纯疱疹病毒胸苷激酶基因的重组质粒中。 （pBR322 / HSV-tk）将LMtk-和RAT-2tk-细胞向TK +表型转化的频率提高了20至40倍。在仅注射少量质粒DNA分子的细胞中，注射线性分子后的转化频率比注射超螺旋分子后高40倍。通过控制注入受体细胞的基因拷贝数，我们可以获得包含单个拷贝或多达50到100个拷贝的可选基因的转化子。转化基因的多个拷贝没有散布在整个宿主基因组中，而是作为连接体整合在宿主染色体的一个或几个位点上。独立的转化体包含不同染色体上的捐赠基因。串联体中基因拷贝的方向不是随机的。而是将这些副本组织为头对尾的串联阵列。通过分析通过共注射两个相同的载体而获得的转化子，除了载体中的一部分被倒置外，我们能够得出结论，头尾结合物主要是通过同源重组产生的。令人惊讶地，在通过注射超螺旋或线性质粒DNA而获得的转化体中发现了这些头尾连接物。即使我们证明培养的哺乳动物细胞含有非常有效地连接两个DNA分子的酶，而不管其末端的序列或拓扑如何，我们也发现甚至线性质粒DNA也通过同源重组被募集到连接体中。

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