Applications of Recombinant Leishmania amazonensis Expressing egfp or the β-Galactosidase Gene for Drug Screening and Histopathological Analysis
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概要
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Leishmania amazonensis recombinants expressing the enhanced green fluorescent protein (egfp) gene or β-galactosidase gene (lacZ) were constructed for drug screening and histopathological analysis. The egfp or lacZ in a leishmanial transfection vector, p6.5, was introduced into L. amazonensis promastigotes, and egfp or lacZ-carrying recombinant L. amazonensis, La/egfp and La/lacZ, respectively, were obtained. Expression of egfp or lacZ in both promastigotes and amastigotes could be clearly visualized by fluorescence microscopy or by light microscopy with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal), respectively. Fluorescence signal and β-galactosidase activity measured by a colorimetric reaction with chlorophenol red β-D-galactopyranoside (CPRG) were well correlated to the numbers of these parasites. The inhibitory concentration (IC50) of a leishmanicidal drug, amphotericin B, in L. amazonensis promastigotes measured using La/egfp and La/lacZ was similar to that measured by conventional methods such as cell counting, thymidine incorporation and colorimetric assay. Furthermore, the fluorescence signal and absorbance of CPRG correlated well with the numbers of La/egfp and La/lacZ amastigotes in macrophages, respectively, suggesting La/egfp and La/lacZ can be a convenient and useful tool for drug screening not only in promastigotes, but also in amastigotes of L. amazonensis. La/lacZ collected from mouse tissues four weeks after the parasite infection were stained well with X-Gal. La/lacZ allowed parasite detection at high sensitivity in the tissues of infected mice and will be useful for following infections in macrophages in vivo. Thus, the marker-transfected Leishmania parasites constructed in this study will be useful for analyses of Leishmania parasites, especially at the intracellular stage.
- 社団法人 日本実験動物学会の論文
- 2003-04-01
著者
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GOTO Yasuyuki
Department of Neurology, School of Medicine, Sao Paulo University
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MATSUMOTO Yasunobu
Laboratory of Global Animal Resource Science, Graduate School of Agricultural and Life Sciences, The
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Okuno Takahiro
Laboraatory Of Global Animal Resource University
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MATSUMOTO Yoshitsugu
Department of Molecular Immunology, Graduate School of Agriculture and Life Sciences, University of
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Goto Yasuyuki
Department Of Electrical And Electronics Engineering Aichi Institute Of Technology
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Goto Yasuyuki
Department Of Molecular Immunology Graduate School Of Agricultural And Life Sciences The University
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Matsumoto Yoshitsugu
Department Of Molecular Immunology School Of Agricultural And Life Sciences University Of Tokyo
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Matsumoto Yoshitsugu
Department Of Molecular Immunology Graduate School Of Agricultural And Life Sciences The University
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OTSUKA Haruki
Laboraatory of Global Animal Resource University
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Matsumoto Yukio
Division Of Nephrology And Metabolism Department Of Internal Medicine Tokai University School Of Med
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Matsumoto Yasunobu
Faculty Of Agriculture The University Of Tokyo
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Matsumoto Yasunobu
Laboratory Of Global Animal Resource Science Graduate School Of Agricultural And Life Sciences Unive
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Takahiro Okuno
Laboraatory of Global Animal Resource University
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