Stable DNA transformation in the obligate intracellular parasite Toxoplasma gondii by complementation of tryptophan auxotrophy.
Stable DNA transformation in the obligate intracellular parasite Toxoplasma gondii by complementation of tryptophan auxotrophy.
复制标题
DOI：
10.1073/pnas.91.12.5508
复制
发表时间：
1994-06
影响因子：
11.1
通讯作者：
L. Sibley;M. Messina;I. Niesman
中科院分区：
文献类型：
--
作者：
L. Sibley;M. Messina;I. Niesman
弓形虫是一种广泛感染脊椎动物宿主的原生动物寄生虫，是免疫功能低下的人类中一种重要的机会性病原体。虽然弓形虫是符合生化和细胞实验方法，其作为细胞内寄生虫成功的分子基础知之甚少。为了提供一个分子遗传分析系统，我们开发了一个稳定的弓形虫DNA转化系统，该系统基于其天然存在的色氨酸缺失的互补。通过表达大肠杆菌trpB基因完成互补，该基因编码色氨酸合成酶（EC 4.2.1.20）的β亚基，该酶催化吲哚和丝氨酸形成色氨酸。通过电穿孔含有trpB基因和弓形虫表面抗原1 （SAG1）基因序列的质粒SAG1/trpB，并选择在吲哚上生长获得转化子。用SAG1/trpB质粒的圆形获得转化子，效率为10(-4)/细胞。圆形或线性SAG1/trpB的转化导致在不同的非同源位点整合到基因组中。Trp+转化体通常包含串联重复的SAG1/trpB质粒拷贝，并且在没有继续选择的情况下是稳定的。色氨酸表型提供了一个显性的可选择标记，允许外来或改变的基因在弓形虫中表达，并促进对细胞内生存重要基因的分子分析。
The protozoan parasite Toxoplasma gondii infects a wide range of vertebrate hosts and is an important opportunistic pathogen in immunocompromised humans. Although Toxoplasma is amenable to both biochemical and cellular experimental approaches, the molecular basis of its success as an intracellular parasite is poorly understood. To provide a system for molecular genetic analyses, we have developed a stable DNA transformation system for Toxoplasma based on complementation of its naturally occurring tryptophan auxotrophy. Complementation was accomplished by expressing the Escherichia coli trpB gene, encoding the beta subunit of tryptophan synthase (EC 4.2.1.20), the enzyme that catalyzes the formation of tryptophan from indole plus serine. Transformants were obtained by electroporation of a plasmid, called SAG1/trpB, containing the trpB gene flanked by Toxoplasma surface antigen 1 (SAG1) gene sequences and selection for growth on indole. Transformants were obtained with circular forms of the SAG1/trpB plasmid with efficiencies of 10(-4) per cell. Transformation with either circular or linear SAG1/trpB resulted in integration into the genome at distinct, nonhomologous sites. Trp+ transformants typically contained tandemly repeated copies of the SAG1/trpB plasmid and were stable in the absence of continued selection. The Trp phenotype provides a dominant selectable marker that should allow expression of foreign or altered genes in Toxoplasma and facilitate molecular analyses of genes important for intracellular survival.