权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Stage analysis on development of Trypanosoma brucei applying GFP as a reporter.

应用 GFP 作为报告基因对布氏锥虫发育进行阶段分析。

基本信息

批准号：
10670244
负责人：
FUKUMA Toshihide
金额：
$ 1.92万
依托单位：
Kurume University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Although the introduction of exogenous DNA into Trypanosoma brucei procyclic forms was comparatively easy, the introduction into the bloodstream forms was very difficult. As a result of using the particle delivery, we succeeded efficiently introducing plasmid vector into the bloodstream forms further than electroporation. Next, we examined GFP expression used as a reporter. Although the fluorescence of wild type GFP was emitted in the procyclic forms, the fluorescence could not be observed in the bloodstream forms: this was most likely due to the wild type GFP has the property of thermal sensitivity (the post-translational modification does not progress at 37℃). Then, we examined the expression of mutant form GFP (EGFP), and it was proven that the intense fluorescence of EGFP was emitted even in the bloodstream forms. In the meantime, in order to clone the gene which peculiarly appears in each life-cycle stages, we constructed plasmid vectors which can express and autonomously replicate in both of procyclic and bloodstream forms. However, these vectors were unexpectedly integrated into chromosome at the high frequency' and the cause is being analyzed. Next, we examined the improvement on drug selection of bloodstream forms, introduced the neomycin resistance gene, to obtain the stable transformant. Although we succeeded in getting the resistant cell by using the step dilution method with 96-well microplate, the efficiency was not so good in which the period in about 2 months is needed. Then, we examined the acquisition of stable transformant using the phleomycin resistance gene and in vivo drug selection system. In the result, drug resistant transformant was efficiently able to be acquired in the short period for the 1〜2 weeks.

虽然外源DNA导入布鲁氏锥虫原环型相对容易，但导入血流型非常困难。由于使用颗粒递送，我们成功地有效地将质粒载体引入血流形态，而不是电穿孔。接下来，我们检查了作为记者使用的GFP表达。野生型GFP虽然以原环形式发出荧光，但在血流形式中无法观察到荧光，这很可能是由于野生型GFP具有热敏性（翻译后修饰在37℃下不进行）。然后，我们检测了突变型GFP （EGFP）的表达，并证明即使在血流形式中也能发出强烈的EGFP荧光。同时，为了克隆每个生命周期阶段特有的基因，我们构建了能够在原循环和血流形式中表达和自主复制的质粒载体。然而，这些载体意外地以高频率整合到染色体中，目前正在分析其原因。接下来，我们考察了血液形式药物选择的改进，引入了新霉素耐药基因，以获得稳定的转化。虽然我们用96孔微孔板阶梯式稀释法获得了耐药细胞，但效率不高，需要2个月左右的时间。然后，我们研究了利用静脉霉素耐药基因和体内药物选择系统获得稳定的转化。结果表明，在1 ~ 2周的短时间内，有效地获得了耐药转化。