DaRocha Wanderson D, Silva Rosiane A, Bartholomeu Daniella C, Pires Simone F, Freitas Jorge M, Macedo Andrea M, Vazquez Martin P, Levin Mariano J, Teixeira Santuza M R
Departamento de Bioquímica e Imunologia, ICB, Universidade Federal de Minas Gerais, MG 31270-010, Belo Horizonte, Brazil.
Parasitol Res. 2004 Jan;92(2):113-20. doi: 10.1007/s00436-003-1004-5. Epub 2003 Nov 21.
To improve transfection efficiency in Trypanosoma cruzi, we developed a new electroporation protocol and expression vectors which use luciferase and green and red fluorescent proteins as reporter genes. In transient transfections, the electroporation conditions reported here resulted in luciferase expression 100 times higher than the levels obtained with previously described protocols. To verify whether sequences containing different trans-splicing signals influence reporter gene expression, we compared DNA fragments corresponding to 5' untranslated plus intergenic (5' UTR plus Ig) regions from GAPDH, TcP2beta, alpha- and beta- tubulin and amastin genes. Vectors containing sequences derived from the first four genes presented similar efficiencies and resulted in luciferase expression in transiently transfected epimastigotes that was up to 10 times higher than that for a control vector. In contrast, the amastin 5' UTR plus Ig resulted in lower levels of reporter gene expression. We also constructed a vector containing an expression cassette designed to be targeted to the tubulin locus of the parasite.
为提高克氏锥虫的转染效率，我们开发了一种新的电穿孔方案和表达载体，其使用荧光素酶以及绿色和红色荧光蛋白作为报告基因。在瞬时转染中，此处报道的电穿孔条件导致荧光素酶表达比先前所述方案获得的水平高100倍。为验证包含不同反式剪接信号的序列是否影响报告基因表达，我们比较了对应于甘油醛-3-磷酸脱氢酶（GAPDH）、TcP2β、α-和β-微管蛋白以及无鞭毛体蛋白基因的5'非翻译区加基因间区（5'UTR加Ig）区域的DNA片段。包含源自前四个基因序列的载体表现出相似的效率，并在瞬时转染的前鞭毛体中导致荧光素酶表达，其比对照载体高10倍。相比之下，无鞭毛体蛋白5'UTR加Ig导致报告基因表达水平较低。我们还构建了一个载体，其包含一个设计用于靶向寄生虫微管蛋白基因座的表达盒。
