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

虽然外源DNA导入前环型布氏锥虫相对容易，但导入血流型布氏锥虫却非常困难。作为使用颗粒递送的结果，我们成功地将质粒载体有效地引入血流形式中，而不是电穿孔。接下来，我们检查了用作报告基因的GFP表达。虽然野生型GFP的荧光以前环形式发射，但在血流形式中不能观察到荧光：这很可能是由于野生型GFP具有热敏性（在37℃下不进行翻译后修饰）。然后，我们检查了突变形式GFP（EGFP）的表达，并且证明了即使在血流形式中也发射EGFP的强荧光。同时，为了克隆在生命周期各阶段特异出现的基因，我们构建了能在前循环和血流两种形式中表达和自主复制的质粒载体。然而，这些载体出乎意料地以高频率整合到染色体中，原因正在分析中。接下来，我们研究了血流形式的药物选择的改进，引入新霉素抗性基因，以获得稳定的药物。采用96孔板分步稀释法虽然获得了抗性细胞，但效果不理想，需要2个月左右的时间。然后，我们使用腐草霉素抗性基因和体内药物选择系统检查了稳定的耐药性的获得。结果，在1 - 2周的短时间内能够有效地获得耐药性的细菌。

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.