Costa Solange dos Santos, de Assis Golim Marjorie, Rossi-Bergmann Bartira, Costa Fabio Trindade Maranhão, Giorgio Selma
Department of Animal Biology, Biology Institute, Universidade Estadual de Campinas Caixa Postal 6109, Cep 13.083-970 Campinas, São Paulo, Brazil.
Korean J Parasitol. 2011 Dec;49(4):357-64. doi: 10.3347/kjp.2011.49.4.357. Epub 2011 Dec 16.
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.
使用编码抗生素抗性基因（如硫酸氨基糖苷遗传霉素，G418）的游离型载体，将绿色荧光蛋白（GFP）导入多种利什曼原虫。大多数关于表达GFP的利什曼原虫的报道使用的是有鞭毛的细胞外前鞭毛体，即沙蝇载体中肠内检测到的寄生虫阶段；而对无鞭毛体（在哺乳动物体内检测到的寄生虫阶段）进行的研究较少。在本研究中，对体外利用G418筛选亚马逊利什曼原虫前鞭毛体和无鞭毛体的效率以及体内利用G418筛选进行了比较。表达GFP的前鞭毛体长时间保留游离型质粒，G418处理对于体外筛选是必要且有效的。相比之下，在没有G418筛选的情况下，表达GFP的无鞭毛体显示出游离型质粒的低保留率，并且在体外对抗生素的敏感性较低。使用感染BALB/c小鼠进行G418筛选的方案也表明，皮肤病变中的无鞭毛体对抗生素的敏感性较低。
