Elert-Dobkowska Ewelina, Hennings J Christopher, Hübner Christian A, Beetz Christian
Department of Clinical Chemistry and Laboratory Diagnostics, Jena University Hospital, 07747 Jena, Germany; Department of Genetics, Institute of Psychiatry and Neurology, 02-957 Warsaw, Poland.
Institute of Human Genetics, Jena University Hospital, 07747 Jena, Germany.
Anal Biochem. 2015 Apr 1;474:35-7. doi: 10.1016/j.ab.2015.01.007. Epub 2015 Jan 20.
Following locus-specific genome editing of mouse embryonic stem cells (ESCs), the identification of correctly targeted clones remains a challenge. We applied multiplex ligation-dependent probe amplification (MLPA) to screen for homologous recombination-based genomic integration of a knockout construct in which part of a gene is deleted. All candidate ESCs thereby identified were subsequently validated by conventional methods. Thus, MLPA represents a highly reliable as well as cost- and time-efficient alternative to currently applied methods such as Southern blotting and polymerase chain reaction (PCR)-based approaches. It is also applicable to knockin recombination strategies and compatible with the CRISPR/Cas9 system and other genome editing strategies.
在对小鼠胚胎干细胞（ESC）进行位点特异性基因组编辑后，鉴定正确靶向的克隆仍然是一项挑战。我们应用多重连接依赖探针扩增（MLPA）来筛选基于同源重组的基因敲除构建体的基因组整合，该构建体中部分基因被删除。由此鉴定出的所有候选ESC随后均通过传统方法进行了验证。因此，MLPA是一种高度可靠且经济高效的替代方法，可替代目前应用的方法，如Southern印迹法和基于聚合酶链反应（PCR）的方法。它也适用于基因敲入重组策略，并与CRISPR/Cas9系统及其他基因组编辑策略兼容。
