Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av do Café, sn/n, 14040-903 Ribeirão Preto, SP, Brazil; Núcleo de Apoio à Pesquisa em Produtos Naturais e Sintéticos, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av do Café, sn/n, 14040-903 Ribeirão Preto, SP, Brazil; Núcleo de Apoio à Pesquisa em Produtos Naturais e Sintéticos, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
Exp Parasitol. 2014 Mar;138:40-7. doi: 10.1016/j.exppara.2014.01.004. Epub 2014 Jan 16.
Neospora caninum is an Apicomplexa parasite related to abortion and losses of fertility in cattle. The amenability of Toxoplasma gondii and Plasmodium to genetic manipulation offers several tools to determine the invasion and replication processes, which support posterior strategies related to the combat of these diseases. For Plasmodium the use of pyrimethamine as an auxiliary drug on malaria treatment has been affected by the rise of resistant strains and the analyses on Dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene indicated several point mutations. In this work we developed a method for stable insertion of genes based on resistance to pyrimethamine. For that, the coding sequence of NcDHFR-TS (Dihydrofolate reductase-thymidylate synthase) was point mutated in two amino acids, generating DHFRM2M3. The DHFRM2M3 flanked by the promoter and 3'UTR of Ncdhfr-ts (Ncdhfr-DHFRM2M3) conferred resistance to pyrimethamine after transfection. For illustration of stability and expression, the cassette Ncdhfr-DHFRM2M3 was ligated to the reporter gene Lac-Z (β-galactosidase enzyme) controlled by the N. caninum tubulin promoter and was transfected and selected in N. caninum. The cassette was integrated into the genome and the selected tachyzoites expressed Lac-Z, allowing the detection of tachyzoites by the CPRG reaction and X-gal precipitation. The obtainment of transgenic N. caninum resistant to pyrimethamine confirms the effects on DHFR-TS among the Apicomplexa members and will support future approaches on pholate inhibitors for N. caninum prophylaxis. The construction of stable tachyzoites based on vectors with N. caninum promoters initiates the molecular manipulation of this parasite independently of T. gondii.
刚地弓形虫是一种与牛流产和繁殖力丧失有关的顶复门寄生虫。疟原虫和刚地弓形虫对遗传操作的易感性为确定入侵和复制过程提供了几种工具，这些过程支持了针对这些疾病的后续策略。对于疟原虫，在疟疾治疗中使用乙胺嘧啶作为辅助药物受到耐药株上升的影响，对二氢叶酸还原酶-胸苷酸合酶（DHFR-TS）基因的分析表明存在几个点突变。在这项工作中，我们开发了一种基于对乙胺嘧啶的抗性的稳定基因插入方法。为此，将 NcDHFR-TS（二氢叶酸还原酶-胸苷酸合酶）的编码序列在两个氨基酸处点突变，产生 DHFRM2M3。侧翼由 Ncdhfr-ts（Ncdhfr-DHFRM2M3）启动子和 3'UTR 组成的 DHFRM2M3 在转染后对乙胺嘧啶产生抗性。为了说明稳定性和表达，将 Ncdhfr-DHFRM2M3 盒连接到由刚地弓形虫微管蛋白启动子控制的报告基因 Lac-Z（β-半乳糖苷酶酶）上，并在刚地弓形虫中进行转染和选择。该盒整合到基因组中，被选择的速殖子表达 Lac-Z，允许通过 CPRG 反应和 X-gal 沉淀检测速殖子。获得对乙胺嘧啶具有抗性的转基因刚地弓形虫证实了顶复门成员中 DHFR-TS 的作用，并将支持未来针对刚地弓形虫预防的叶酸抑制剂的方法。基于带有刚地弓形虫启动子的载体构建稳定的速殖子，独立于刚地弓形虫开始了对这种寄生虫的分子操作。
