Korean J Parasitol.  2011 Dec;49(4):357-364. 10.3347/kjp.2011.49.4.357.

Use of In Vivo and In Vitro Systems to Select Leishmania amazonensis Expressing Green Fluorescent Protein

• Costa SD
• de Assis Golim M
• Rossi-Bergmann B
• Costa FT
• Giorgio S

• KMID: 1715740
• DOI: http://doi.org/10.3347/kjp.2011.49.4.357

Abstract

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.

Keyword

Leishmania amazonensis; green fluorescent protein; macrophage; geneticin

MeSH Terms

Amebicides/*pharmacology
Animals
Flow Cytometry
Gentamicins/*pharmacology
Green Fluorescent Proteins/*chemistry
Host-Parasite Interactions
Leishmania mexicana/drug effects/genetics/*growth & development
Leishmaniasis, Cutaneous/*parasitology
Luminescent Agents/*chemistry
Macrophages, Peritoneal/parasitology
Mice
Mice, Inbred BALB C
Organisms, Genetically Modified
Spectrometry, Fluorescence
Amebicides
Gentamicins
Luminescent Agents
Green Fluorescent Proteins