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首页> 外文期刊>Molecular and Cellular Biology >The Mechanism of Mammalian Gene Replacement Is Consistent with the Formation of Long Regions of Heteroduplex DNA Associated with Two Crossing-Over Events
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The Mechanism of Mammalian Gene Replacement Is Consistent with the Formation of Long Regions of Heteroduplex DNA Associated with Two Crossing-Over Events

机译：哺乳动物基因替换的机制与两个交叉事件相关的异源双链DNA长区域的形成一致。

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In this study, the mechanism of mammalian gene replacement was investigated. The system is based on detecting homologous recombination between transferred vector DNA and the haploid, chromosomal immunoglobulin μ-δ region in a murine hybridoma cell line. The backbone of the gene replacement vector (pCμCδpal) consists of pSV2neo sequences bounded on one side by homology to the μ gene constant (Cμ) region and on the other side by homology to the δ gene constant (Cδ) region. The Cμ and Cδ flanking arms of homology were marked by insertions of an identical 30-bp palindrome which frequently escapes mismatch repair when in heteroduplex DNA (hDNA). As a result, intermediates bearing unrepaired hDNA generate mixed (sectored) recombinants following DNA replication and cell division. To monitor the presence and position of sectored sites and, hence, hDNA formation during the recombination process, the palindrome contained a unique NotI site that replaced an endogenous restriction enzyme site at each marker position in the vector-borne Cμ and Cδ regions. Gene replacement was studied under conditions which permitted the efficient recovery of the product(s) of individual recombination events. Analysis of marker segregation patterns in independent recombinants revealed that extensive hDNA was formed within the Cμ and Cδ regions. In several recombinants, palindrome markers in the Cμ and Cδ regions resided on opposite DNA strands (trans configuration). These results are consistent with the mammalian gene replacement reaction involving two crossing-over events in homologous flanking DNA.
机译：在这项研究中，研究了哺乳动物基因替换的机制。该系统基于检测鼠杂交瘤细胞系中转移的载体DNA与单倍体，染色体免疫球蛋白μ-δ区之间的同源重组。基因替换载体（pCμCδpal）的骨架由pSV2neo序列组成，pSV2neo序列的一侧与μ基因常数（Cμ）区域同源，而另一侧与δ基因常数（Cδ）区域同源。同源的Cμ和Cδ侧臂的特征是插入相同的30 bp回文，当在异源双链DNA（hDNA）中时经常逃脱错配修复。结果，携带未修复hDNA的中间体在DNA复制和细胞分裂后产生混合（扇形）重组体。为了监测扇形位点的存在和位置以及重组过程中hDNA的形成，回文包含一个独特的 Not I位点，该位点取代了载体中每个标记位点的内源性限制酶位点，包含Cμ和Cδ区。在允许有效回收单个重组事件产物的条件下研究了基因置换。独立重组体中标记分离模式的分析表明，广泛的hDNA在Cμ和Cδ区域内形成。在一些重组体中，Cμ和Cδ区的回文标记位于相反的DNA链上（ trans 构型）。这些结果与哺乳动物基因置换反应一致，该基因置换反应涉及同源侧翼DNA中的两个交换事件。

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