Molecular Dissection of Early Toxoplasma gondii Bradyzoite Differentiation

描述（由申请方提供）：弓形虫是一种人畜共患寄生虫，分布于世界各地。免疫抑制患者中的疾病主要是由于休眠的缓殖子包囊的重新激活。缓殖子形式的发育可以在体外研究，但很少有分子或遗传学的细节，这一重要的过程是已知的。利用一种新的多功能微阵列，下面概述的研究将提供一个全面的全基因组分析缓殖子的形成。根据迄今为止产生的初步数据，我们假设，非常早期的基因产物是需要的缓殖子分化的启动，这将通过有针对性的转基因工程和/或敲入寄生虫进行研究。具体目标1：确定缓殖子诱导的基因。1.a.定义假定的缓殖子基因的表达模式。将采用多种诱导方法来确定独特的缓殖子特异性基因。1.b.关注下游基因靶向。使用合理的选择过程加上生物信息学分析，基因将被靶向用于基因敲除实验。具体目标2：通过遗传扰动对关键阶段特异性转录本进行分子解剖。2.a.基因删除（敲除）选定的目标基因。电转染和药物选择将用于产生等位基因敲除突变体。2.b.体外评估假定的缓殖子诱导突变体。在体外缓殖子形成的多种方法中有缺陷的突变体将鉴定可能在分化过程中必不可少的基因。2.c.通过适合性测定和互补验证靶基因对缓殖子诱导的特异性影响。为了确保表型与缓殖子形成直接相关，将补充突变体并验证其生长和感染适应性。这个项目的长期目标是建立一个更完整的了解普遍存在的和致病性的T。刚地。本研究中提出的实验有望鉴定出一些参与T.刚地。除了从一般生物学的角度来看是感兴趣的，这样的基因应该提供新的见解缓殖子特异性治疗的发展。除了提高我的研究技能，这可能会提供关键的见解，导致开发针对缓殖子靶点的药物，或用于开发疫苗的疾病减毒生物。

DESCRIPTION (provided by applicant): Toxoplasma gondii is a zoonotic human parasite with worldwide distribution. Disease in the growing ranks of immunosuppressed patients is primarily due to reactivation of dormant bradyzoite cysts. Development of the bradyzoite form can be studied in vitro, but few molecular or genetic details underlying this important process are known. Taking advantage of a novel multifunctional microarray, the research outlined below will provide a comprehensive genome-wide analysis of bradyzoite formation. Based on preliminary data generated to date, we hypothesize that very early gene products are required for the initiation of bradyzoite differentiation, which will be studied through targeted engineering of transgenic and/or knock-put parasites. Specific Aim 1: Identification of genes that define bradyzoite induction. 1.a. Define expression patterns for putative bradyzoite genes. Multiple induction methods will be employed to ascertain unique bradyzoite-specific genes. 1.b. Focus downstream gene targeting. Using a rational selection process coupled with bioinformatic analysis, genes will be targeted for genetic knock-out experiments. Specific Aim 2: Molecular dissection of critical stage-specific transcripts through genetic perturbation. 2.a. Genetically delete (knock-out) selected genes of interest. Electro-transfection and drug selection will be used to generate allelic knock-out mutants. 2.b. Assess putative bradyzoite induction mutants in vitro. Mutants defective in multiple methods of in vitro bradyzoite formation will identify genes likely to be essential in the differentiation process. 2.c. Verify specific influence of the target gene on bradyzoite induction through fitness assays and complementation. To ensure that phenotypes are directly related to bradyzoite formation, mutants will be complemented and verified for growth and infectivity fitness. The long-term goal of this project is to establish a more complete understanding of the ubiquitous and pathogenic bradyzoite stage of T. gondii. The experiments proposed in this study are expected to lead to the identification of a number of genes involved in the differentiation process in T. gondii. Besides being of interest from a general biological standpoint, such genes should provide novel insights into the development of bradyzoite-specific therapeutics. In addition to increasing my research skills, this may provide key insights leading to the development of drugs designed against bradyzoite targets, or disease-attenuated organisms useful in developing vaccines.