Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.
Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.
复制标题
当引入培养的小鼠细胞时，含有小鼠 rDNA 的质粒不会与细胞核糖体基因重组。
DOI：
10.1128/mcb.4.4.576-582.1984
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发表时间：
1984
影响因子：
5.3
通讯作者：
Reeder,RH
中科院分区：
文献类型：
--
作者：
Steele,RE;Bakken,AH;Reeder,RH
我们研究了含有小鼠rDNA重复片段的质粒通过转染进入培养的小鼠细胞后的命运。除了rDNA片段外，质粒还含有单纯疱疹病毒1型胸苷激酶基因，以便在转染胸苷激酶缺陷小鼠细胞后对质粒进行选择。到目前为止，转染的质粒dna与宿主细胞序列之间的同源重组尚未被记录。我们认为，rRNA基因在小鼠基因组中的高重复频率（每个二倍体细胞200个拷贝）可能为含有rDNA的质粒与宿主细胞rDNA序列之间获得同源重组事件创造了有利条件。质粒在存在和不存在载体DNA的情况下被引入细胞，并以共价闭合的环状和线性分子的形式引入细胞。通过DNA限制性酶切和杂交、分子克隆和核分离等方法，对不同细胞系基因组中的质粒整合位点进行了检测。在检查的7个细胞系中，没有证据表明质粒已经整合到细胞的rRNA基因簇中。因此，引入哺乳动物细胞的克隆dna明显缺乏位点特异性整合，似乎并不仅仅是由于大多数宿主细胞序列所呈现的小靶标。
We have examined the fate of plasmids containing a segment of a mouse rDNA repeat after they were introduced by transfection into cultured mouse cells. In addition to the rDNA segment, the plasmids contained the thymidine kinase gene from herpes simplex virus 1 to allow for selection of the plasmid after transfection into thymidine kinase-deficient mouse cells. Thus far, no cases of homologous recombination between transfected plasmid DNAs and host cell sequences have been documented. We reasoned that the high repetition frequency of the rRNA genes in the mouse genome (200 copies per diploid cell) might create a favorable situation for obtaining homologous recombination events between the plasmids containing rDNA and host cell rDNA sequences. The plasmids were introduced into cells in both the presence and the absence of carrier DNA and both as covalently closed circles and linear molecules. The sites of plasmid integration in the genomes of various cell lines were examined by DNA restriction digests and hybridization, molecular cloning, and nuclear fractionation. In the seven cell lines examined, there was no evidence that the plasmids had integrated into the rRNA gene clusters of the cell. Thus, the apparent absence of site-specific integration of cloned DNAs introduced into mammalian cells does not appear to be due simply to the small target presented by most host cell sequences.