Use of In Vivo and In Vitro Systems to Select Leishmania amazonensis Expressing Green Fluorescent Protein
Use of In Vivo and In Vitro Systems to Select Leishmania amazonensis Expressing Green Fluorescent Protein
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DOI：
10.3347/kjp.2011.49.4.357
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发表时间：
2011-12-01
影响因子：
--
通讯作者：
Giorgio, Selma
中科院分区：
文献类型：
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作者：
Costa, Solange dos Santos;Golim, Marjorie de Assis;Giorgio, Selma
利用编码抗生素耐药基因的上体载体，如氨基糖苷类基因硫酸盐(G418)，利用绿色荧光蛋白(GFP)对各种利什曼原虫进行了基因工程。大多数关于GFP-利什曼原虫的报道都使用了有鞭毛的胞外前鞭毛体，这是在沙蝇媒介的中肠中检测到的寄生虫阶段；较少的研究是用无鞭毛虫进行的，这是在哺乳动物中检测到的寄生虫阶段。本研究比较了绿色荧光蛋白-亚马逊利什曼原虫前鞭毛体和无鞭毛体G418体外选择的效率和体内G418选择的效率。GFP-前鞭毛体可以较长时间地保留上体质粒，G418处理是进行体外筛选的必要和有效的方法。相反，在没有G418选择的情况下，GFP-无鞭毛体显示出低保留率和体外对抗生素的低敏感性。使用感染的BALB/c小鼠进行G418选择的方案也表明，对皮肤病变中的无鞭毛虫的抗生素敏感性较低。
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.