Transcriptome analysis for tunic cells of the ascidian, Ciona intestinalis

海鞘、玻璃海鞘被膜细胞的转录组分析

基本信息

批准号：
22510209
负责人：
KAWASHIMA Takeshi
金额：
$ 2.91万
依托单位：
沖縄科学技術大学院大学 (2011-2012)Okinawa Institute of Science and Technology (2010)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2010
资助国家：
日本
起止时间：
2010 至 2012
项目状态：
已结题

项目摘要

This study intend to build a molecular biological basis of tunic cells in an ascidian, Ciona intestinalis, which is now an important model organism of EvoDevo research. Although tunic is the main characteristic-organ of this animal, the group of cells which are inlaying of the organ is not really investigated to date.The original plan of this study was made up of two parts of strategy that are (1) decoding of RNAs expressed in the tunic cells and, (2) molecular and cellular study for the tunic cells. The RNA extraction from tunic cells for NGS sequencing were difficult than I have been expected.To avoid this problem, we corrected the original plan in the term as followings; (a) we collected the frozen sample of tunic-organ, (b) we provided the continued requirement-study to isolate the RNAs from tunic cells and (c) we designed a custom microarray for future analysis of gene expression in tunic cells. (See Matsumae et al. 2013 in Section 13.)In order to forward the second of abovestrategies , we were concerned about the cellulose filament which is the main component of tunic as an obstruction for analysis of tunic cells. On the advices of Prof. Hirose who is one of cooperative researcher of this study, we overcame thissecond difficulty. We established a method to isolate the tunic cells from the tunic-organ by means of incubation of tunic with a glass slide. This method allowed us to observe a clear view of the tunic-cells and its nuclei by DAPI-staining. We have verified that two types of tunic-cells are isolated from the crude tunic that contains at least three types of cells by our method. We expect that this method has made possible for variety of analytical approaches including of in situ hybridization and cell culture of C. intestinalis.As for future perspectives, because we have collected the tunic-organ sample for RNA-extraction, I'm planning for decoding the RNA sequences of tunic cells within around next two years.

这项研究旨在在海外塞奥纳肠中膜细胞的分子生物学基础，这是现在的重要模型生物。尽管外衣是该动物的主要特征 - 轨道，但迄今为止并未真正研究过器官的细胞组。这项研究的原始计划由策略的两个部分组成（1）在外束细胞中表达的RNA和（2）外束细胞的分子和细胞研究。从外衣细胞中提取NGS测序的RNA比我预期的要困难。为避免此问题，我们在以下任期内纠正了原始计划。（a）我们收集了束缚孔管的冷冻样品，（b）我们提供了持续的需求研究，以将RNA与外束细胞分离，（c）我们设计了一种自定义微阵列，用于将未来的基因表达分析中衣细胞中分析。（请参阅Matsumae等人，2013年的第13节。）为了转发第二个Abovestrategies，我们担心纤维素细丝，纤维素丝是上衣的主要组成部分，是对束缚细胞分析的障碍物。在这项研究的合作研究人员之一的Hirose教授的建议下，我们克服了这一方面的困难。我们建立了一种方法，通过将上衣与载玻片孵育，将外衣细胞与外衣孔分离。这种方法使我们能够通过DAPI染色观察束腰外衣及其核的视野。我们已经验证了两种类型的外衣细胞是通过我们的方法中至少包含三种细胞的粗上衣中分离出来的。我们预计，这种方法已成为多种分析方法，包括原位杂交和肠道梭菌的细胞培养。