Pakpour Nazzy, Cheung Kong Wai, Souvannaseng Lattha, Concordet Jean-Paul, Luckhart Shirley
Department of Medical Microbiology and Immunology, University of California, Davis, USA.
J Vis Exp. 2010 Dec 26(46):2355. doi: 10.3791/2355.
Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development (1). From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in (2)). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice (3,4). We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.
疟原虫是疟疾的病原体，通过受感染的按蚊叮咬传播，每年导致超过2.5亿例新感染。尽管经过数十年的研究，仍然没有针对疟疾的疫苗，这凸显了开发新型控制策略的必要性。一种创新方法是利用基因改造蚊子来有效控制疟原虫传播。通过靶向诱变对蚊子细胞信号通路进行有意改变，已被发现可调节寄生虫发育（1）。从这些研究中，我们可以开始识别潜在的转化基因靶点。传统上，靶向诱变依赖于目标基因与大型DNA分子之间的同源重组。然而，构建和使用这种复杂的DNA分子来生成稳定转化的细胞系成本高昂、耗时且往往效率低下。因此，使用锁核酸修饰的寡核苷酸（LNA-ONs）的策略为将人工单核苷酸替换引入游离和染色体DNA基因靶点提供了一种有用的替代方法（综述见（2））。LNA-ON介导的靶向诱变已被用于在酵母和小鼠的培养细胞中对感兴趣的基因引入点突变（3,4）。我们在此表明，LNA-ONs可用于在转染的游离靶点中引入单核苷酸变化，从而在冈比亚按蚊和斯氏按蚊细胞中导致从蓝色荧光蛋白（BFP）表达切换到绿色荧光蛋白（GFP）表达。这种转换首次证明了LNA-ONs可介导蚊子细胞中目标基因的有效诱变，并表明该技术可能适用于体外和体内染色体靶点的诱变。
