Department of Ecological Developmental Adaptability Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan.
Research Fellow (DC2) of Japan Society for the Promotion of Science, Tokyo, Japan.
Dev Growth Differ. 2021 Apr;63(3):189-198. doi: 10.1111/dgd.12721.
The ability to manipulate gene expression at a specific region in a tissue or cell culture system is critical for analysis of target gene function. For chick embryos/cells, several gene introduction/induction methods have been established such as those involving retrovirus, electroporation, sonoporation, and lipofection. However, these methods have limitations in the accurate induction of localized gene expression. Here we demonstrate the effective application of a recently developed light-dependent gene expression induction system (LightOn system) using the Neurospora crassa photoreceptor Vivid fused with a Gal4 DNA binding domain and p65 activation domain (GAVPO) that alters its activity in response to light stimulus in a primary chicken cell culture system. We show that the gene expression level and induction specificity in this system are strongly dependent on the light irradiation conditions. Especially, the irradiation interval is an important parameter for modulating gene expression; for shorter time intervals, higher induction specificity can be achieved. Further, by adjusting light irradiation conditions, the expression level in primary chicken cells can be regulated in a multiple step manner, in contrast to the binary expression seen for gene disruption or introduction (i.e., null or overexpression). This result indicates that the light-dependent expression control method can be a useful technique in chick models to examine how gene function is affected by gradual changes in gene expression levels. We applied this light induction system to regulate Sox9 expression in cultures of chick limb mesenchyme cells and showed that induced SOX9 protein could modulate expression of downstream genes.
在组织或细胞培养系统中特定区域操纵基因表达的能力对于分析靶基因功能至关重要。对于鸡胚胎/细胞，已经建立了几种基因导入/诱导方法，例如涉及逆转录病毒、电穿孔、声穿孔和脂质体转染的方法。然而，这些方法在精确诱导局部基因表达方面存在局限性。在这里，我们展示了一种最近开发的光依赖性基因表达诱导系统（LightOn 系统）在原代鸡细胞培养系统中的有效应用，该系统使用 Neurospora crassa 光受体 Vivid 融合了 Gal4 DNA 结合域和 p65 激活域（GAVPO），该系统根据光刺激改变其活性。我们表明，该系统中的基因表达水平和诱导特异性强烈依赖于光照射条件。特别是，照射间隔是调节基因表达的重要参数；较短的时间间隔可以实现更高的诱导特异性。此外，通过调整光照射条件，可以以多步方式调节原代鸡细胞中的表达水平，与基因缺失或引入（即 null 或过表达）的二元表达形成对比。这一结果表明，光依赖性表达控制方法可以成为研究基因功能如何受基因表达水平逐渐变化影响的鸡模型中的有用技术。我们将该光诱导系统应用于调节鸡肢间充质细胞培养物中的 Sox9 表达，结果表明诱导的 SOX9 蛋白可以调节下游基因的表达。
