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

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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.