Functional Analysis of Large Plasmodium falciparum genes

描述(由申请人提供)：近年来，通过对恶性疟原虫和其他疟原虫物种进行基因组测序，疟疾研究的进展大大加快。将系统生物学工具应用于基因组数据，为药物和疫苗开发确定了新的途径和目标。确定这些靶点潜力的一个关键组成部分是分析相关基因(S)的功能，并确定干扰对该功能的影响。恶性疟原虫不适用于传统的遗传方法，因此功能测试是通过引入定向突变来实现的，方法是用构建的质粒体进行转基因。随着基因组测序工作的进行，恶性疟原虫的转基因方法的发展已经取得了重大进展，并产生了许多关键的见解。然而，到目前为止，还没有方法可以在不破坏基因、互补、等位基因替换或对长基因的中心区进行突变的情况下删除内部序列，如var基因。Var基因家族编码PfEMP1蛋白，介导细胞黏附和抗原变异。目前分析PfEMP1粘附性的方法依赖于测试分离的结构域，当结构域是大型蛋白质的一部分时，数据可能不能代表功能。PfEMP1在疟疾发病机制中的重要性使得设计长基因中心区的遗传分析方法变得至关重要。我们在这里提出了一种方法，在不破坏表达的情况下将序列整合到恶性疟原虫长基因的中心区，从而可以对关键基因的基因功能进行详细分析。

DESCRIPTION (provided by applicant): Progress in malaria research has been substantially accelerated in recent years by genome sequencing of P. falciparum and other Plasmodium species. Application of systems biology tools to the genome data has identified new pathways and targets for drug and vaccine development. A critical component of confirming the potential of such targets is analyzing the function of the relevant gene(s) and determining the effect of interference with that function. P. falciparum is not amenable to traditional genetic approaches so function is tested by the introduction of directed mutations by transfection with plasmid constructs. There have been major strides in development of transfection methods for P. falciparum, hand-in-hand with the genome sequencing effort, and many critical insights have resulted. However, as yet there are no methods that allow deletion of internal sequences without gene disruption, complementation, allelic replacement, or mutagenesis for the central regions of long genes, like var genes. The var gene family encodes PfEMP1 proteins, which mediate cytoadherence and antigenic variation. Current methods for analysis of PfEMP1 adhesive properties rely on testing isolated domains and the data may not be representative of function when the domain is part of a large protein. The importance of PfEMP1 to malaria pathogenesis makes it critical to devise methods for genetic analysis of central regions of long genes. We here propose a method to integrate sequences into central regions of long P. falciparum genes without ablation of expression, allowing detailed analysis of gene function for critically important genes.