Cooper R, de Jesus A R, Cross G A
Laboratory of Molecular Parasitology, Rockefeller University, New York 10021.
J Cell Biol. 1993 Jul;122(1):149-56. doi: 10.1083/jcb.122.1.149.
Null mutants of the Trypanosoma cruzi insect stage-specific glycoprotein GP72 were created by targeted gene replacement. Targeting plasmids were constructed in which the neomycin phosphotransferase and hygromycin phosphotransferase genes were flanked by GP72 sequences. These plasmids were sequentially transfected into T. cruzi epimastigotes by electroporation. Southern blot analyzes indicated that precise replacement of the two genes had occurred. No aberrant rearrangements occurred at the GP72 locus and no GP72 gene sequences had been translocated elsewhere in the genome. Western blots confirmed that GP72 is not expressed in these null mutants. The morphology of the mutants is dramatically different from wild-type. In both mutant and wild-type parasites, the flagellum emerges from the flagellar pocket. In the null mutant the normal attachment of the flagellum to the cell membrane of the parasite is lost.
通过靶向基因替换构建了克氏锥虫昆虫阶段特异性糖蛋白GP72的缺失突变体。构建了靶向质粒，其中新霉素磷酸转移酶和潮霉素磷酸转移酶基因两侧为GP72序列。通过电穿孔将这些质粒依次转染到克氏锥虫前鞭毛体中。Southern印迹分析表明两个基因已发生精确替换。在GP72基因座未发生异常重排，且GP72基因序列未转移至基因组的其他位置。Western印迹证实这些缺失突变体中不表达GP72。突变体的形态与野生型有显著差异。在突变体和野生型寄生虫中，鞭毛均从鞭毛囊中伸出。在缺失突变体中，鞭毛与寄生虫细胞膜的正常附着丧失。
