Kawaguchi Akane, Utsumi Nanami, Morita Maki, Ohya Aya, Wada Shuichi
Department of Bioscience, Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, Japan.
Genesis. 2015 Jan;53(1):170-82. doi: 10.1002/dvg.22834. Epub 2014 Nov 13.
Temporally controlled induction of gene expression is a useful technique for analyzing gene function. To make such a technique possible in Ciona intestinalis embryos, we employed the cis-regulatory region of the heat-shock protein 70 (HSP70) gene Ci-HSPA1/6/7-like for heat-inducible gene expression in C. intestinalis embryos. We showed that Ci-HSPA1/6/7-like becomes heat shock-inducible by the 32-cell stage during embryogenesis. The 5'-upstream region of Ci-HSPA1/6/7-like, which contains heat-shock elements indispensable for heat-inducible gene expression, induces the heat shock-dependent expression of a reporter gene in the whole embryo from the 32-cell to the middle gastrula stages and in progressively restricted areas of embryos in subsequent stages. We assessed the effects of heat-shock treatments in different conditions on the normality of embryos and induction of transgene expression. We evaluated the usefulness of this technique through overexpression experiments on the well-characterized, developmentally relevant gene, Ci-Bra, and showed that this technique is applicable for inferring the gene function in C. intestinalis.
基因表达的时间控制诱导是分析基因功能的一项有用技术。为了使这种技术在玻璃海鞘胚胎中成为可能，我们利用热休克蛋白70（HSP70）基因Ci-HSPA1/6/7-like的顺式调控区域，在玻璃海鞘胚胎中进行热诱导基因表达。我们发现Ci-HSPA1/6/7-like在胚胎发育的32细胞阶段变得对热休克有诱导性。Ci-HSPA1/6/7-like的5'上游区域包含热诱导基因表达所必需的热休克元件，它能在从32细胞阶段到原肠胚中期的整个胚胎中，以及在随后阶段胚胎中逐渐受限的区域诱导报告基因的热休克依赖性表达。我们评估了不同条件下热休克处理对胚胎正常性和转基因表达诱导的影响。我们通过对特征明确的、与发育相关的基因Ci-Bra进行过表达实验，评估了该技术的实用性，并表明该技术适用于推断玻璃海鞘中的基因功能。
