Delgadillo M G, Liston D R, Niazi K, Johnson P J
Department of Microbiology and Immunology, School of Medicine, Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
Proc Natl Acad Sci U S A. 1997 Apr 29;94(9):4716-20. doi: 10.1073/pnas.94.9.4716.
We have developed methods to transiently and selectably transform the human-infective protist Trichomonas vaginalis. This parasite, a common cause of vaginitis worldwide, is one of the earlier branching eukaryotes studied to date. We have introduced three heterologous genes into T. vaginalis by electroporation and have used the 5' and 3' untranslated regions of the endogenous gene alpha-succinyl CoA synthetase B (alpha-SCSB) to drive transcription of these genes. Transient expression of two reporter proteins, chloramphenicol acetyltransferase (CAT) or luciferase, was detected when electroporating in the presence of 50 microg closed-circular construct. Optimal levels of expression were observed using approximately 2.5 x 10(8) T. vaginalis cells and 350 volts, 960 microFd for electroporation; however, other conditions also led to significant reporter gene expression. A time course following the expression of CAT in T. vaginalis transient transformants revealed the highest level of expression 8-21 hr postelectroporation and showed that CAT activity is undetectable using TLC by 99 hr postelectroporation. The system we established to obtain selectable transformants uses the neomycin phosphotransferase (neo) gene as the selectable marker. Cells electroporated with 20 microg of the NEO construct were plated in the presence of 50 microg/ml paromomycin and incubated in an anaerobic chamber. The paromomycin-resistant colonies that formed within 3-5 days were cultivated in the presence of drug and DNA was isolated for analyses. The NEO construct was shown to be maintained episomally, as a closed-circle, at between 10-30 copies per cell. The ability to transiently and selectably transform T. vaginalis should greatly enhance research on this important human parasite.
我们已经开发出了可对感染人类的原生生物阴道毛滴虫进行瞬时且选择性转化的方法。这种寄生虫是全球阴道炎的常见病因之一，是迄今为止研究的较早分支的真核生物之一。我们通过电穿孔法将三个异源基因导入阴道毛滴虫，并利用内源性基因α-琥珀酰辅酶A合成酶B（α-SCSB）的5'和3'非翻译区来驱动这些基因的转录。当在50微克闭环构建体存在的情况下进行电穿孔时，检测到了两种报告蛋白氯霉素乙酰转移酶（CAT）或荧光素酶的瞬时表达。使用约2.5×10⁸个阴道毛滴虫细胞和350伏、960微法进行电穿孔时观察到了最佳表达水平；然而，其他条件也导致了显著的报告基因表达。对阴道毛滴虫瞬时转化体中CAT表达的时间进程研究表明，电穿孔后8至21小时表达水平最高，并且显示到电穿孔后99小时时，通过薄层层析法无法检测到CAT活性。我们建立的用于获得可选择转化体的系统使用新霉素磷酸转移酶（neo）基因作为选择标记。用20微克NEO构建体进行电穿孔的细胞接种在含有50微克/毫升巴龙霉素的培养基中，并在厌氧箱中培养。在3至5天内形成的抗巴龙霉素菌落用药物培养，并分离DNA进行分析。结果表明，NEO构建体以闭环形式作为附加体在每个细胞中以10至30个拷贝的数量维持。对阴道毛滴虫进行瞬时且选择性转化的能力应会极大地促进对这种重要人类寄生虫的研究。
