Development of highly effective gene knock-in procedure using neural crest-specific gene expression machinery

为了确定神经脊细胞特异性增强子区域以及与该增强子区域结合的转录因子，我们利用内皮素A受体基因座上的几个上游启动子区域进行了荧光素酶检测。因此，我们发现了大约30个碱基作为一个关键的增强子序列，它由两个转录因子的推测串联结合位点组成。在转基因小鼠体内的实验表明，该增强子序列不足以强制表达LacZ基因。这些结果表明，可能需要与其他区域的协调才能在神经脊细胞中正确表达基因。腺病毒转导受精卵对其后来的正常发育具有毒性，而LacZ的强表达被观察到。因此，病毒毒性这一重要问题仍有待克服。

To identify the neural crest cell-specific enhancer region as well as transcription factors that bind to the enhancer region, we conducted luciferase assay using several upstream promoter regions on endothelin type-A receptor gene locus. Consequently, we found that about 30 bases as a critical enhancer sequence that consisted of presumable tandem binding sites of two transcription factors. In vivo experiments in transgenic mice exhibited an insufficiency of this enhancer sequence for neural crest cell-specific forced expression of LacZ gene. These results suggest that a possible coordination with other region may be required to proper gene expression in neural crest cells.Adenoviral transduction in fertilized eggs exhibited toxicity for their later normal development, whereas strong expression of LacZ was observed. Therefore, the important issue of viral toxicity remained to be overcome.