Uemura Shun, Nagaoka Tadahiro, Yokoyama Minesuke, Igarashi Michihiro, Kishi Masashi
Laboratory of Molecular Neuroimaging, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata 951-8510, Japan.
Department of Comparative and Experimental Medicine, Brain Research Institute, Niigata University, Niigata 951-8510, Japan.
Neurosci Res. 2014 Mar;80:91-4. doi: 10.1016/j.neures.2013.11.007. Epub 2013 Dec 27.
Because genetic manipulation occasionally disrupts the expression of the neighboring genes, the chromosomal locus where the transgene has been integrated should be identified in the use of transgenic organisms. By using a new blend of thermostable DNA polymerase, we established a highly efficient method of inverse polymerase chain reaction for this purpose. By using this protocol, we successfully determined the vector integration sites of 2 mouse lines, NSE-tTA and tetO-Cre, the combination of which is a useful tool in neuroscience research. On the basis of this information, we quantified the relative expression amount of the chromosomal genes adjacent to these transgenes and found that the insertion of the tetO-Cre vector significantly altered the mRNA level of one of the examined genes. Considering the potential risk of the insertion effect, we recommend that the vector integration sites of any transgenic lines should be determined routinely by using this method, and that the expression levels of their neighboring genes should be determined.
由于基因操作偶尔会干扰邻近基因的表达，因此在使用转基因生物时，应确定转基因整合的染色体位点。通过使用一种新型热稳定DNA聚合酶混合物，我们为此建立了一种高效的反向聚合酶链反应方法。通过使用该方案，我们成功确定了2个小鼠品系NSE-tTA和tetO-Cre的载体整合位点，它们的组合是神经科学研究中的一种有用工具。基于这些信息，我们对这些转基因相邻的染色体基因的相对表达量进行了定量，发现tetO-Cre载体的插入显著改变了其中一个检测基因的mRNA水平。考虑到插入效应的潜在风险，我们建议应常规使用该方法确定任何转基因品系的载体整合位点，并确定其邻近基因的表达水平。
