Experimental genetic crosses for malaria research

疟疾研究的实验性基因杂交

• 批准号: 10216643
• 负责人: Ashley M Vaughan
• 金额: $ 30.8万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10216643
• 关键词: Acetoacetates; Achievement; Anopheles Genus; Artemisinins; Blood; Cell Death; Cessation of life; Chimerism; Chloroquine resistance; Chromosome Mapping; Cloning; Collaborations; Coupled; Culicidae; Data Set; Drug resistance; Drug usage; Ensure; Erythrocytes; Ethics; Excision; Experimental Genetics; Feeds; Gene Frequency; Generations; Genes; Genetic; Genetic Crosses; Genetic Determinism; Genotype; Grant; Health; Hepatocyte; Human; Hydrolase; Immunocompromised Host; In Vitro; Infection; Infusion procedures; Intervention; Laboratories; Life Cycle Stages; Link; Liver; Malaria; Maps; Modeling; Mus; Pan Genus; Parasites; Pharmaceutical Preparations; Phenotype; Plasmodium falciparum; Point Mutation; Population; Process; Quantitative Trait Loci; Recombinants; Research; Research Project Grants; Salivary Glands; Sampling; Speed; Sporozoites; System; Technology; Time; United States National Institutes of Health; Work; asexual; dietary supplements; experience; genome sequencing; genomic locus; mouse model; success; whole genome

ABSTRACT A concrete way to map genotypes that cause drug resistance phenotypes is by performing a well thought out experimental genetic cross between drug resistance and drug susceptible Plasmodium falciparum strains, isolating recombinant progeny and then using quantitative trait loci mapping to link genotype to phenotype. Three P. falciparum genetic crosses were carried out in splenectomized chimpanzees over 23 years and one of the great achievements of these crosses was the genetic determinant of chloroquine resistance – point mutations in the pfcrt gene. The NIH has now banned chimpanzee research for financial and ethical reasons but we have developed a human-liver chimeric mouse model (the FRG huHep mouse) to replace the chimpanzee for the generation of recombinant progeny from P. falciparum genetic crosses. The FRG mouse lacks the fumaryl acetoacetate hydrolase gene (F designation) and this causes hepatocyte cell death. However, hepatocyte death is controlled with the drug nitisinone. Since only mouse hepatocytes lack fumaryl acetoacetate hydrolase, this enables repopulation of the mouse with human hepatocytes over time with on-off drug use to control the death of mouse hepatocytes and their replacement with human hepatocytes. In close collaboration with the Yecuris Corporation, who creates the FRG huHep mouse, we ensure that the mice we use for our studies have maximal human hepatocyte chimerism and are susceptible to P. falciparum sporozoite infection. Additionally, the mice are able to maintain a human red blood cell (huRBC) population after huRBC infusion and this allows for P. falciparum liver stage-to-blood stage transition in the mouse. Following blood removal, the in vitro expansion of asexual P. falciparum blood stages allows for downstream cloning and then –omics analyses and phenotypic analyses of recombinant progeny. We have already demonstrated our ability to use the FRG huHep/huRBC mouse for the generation of recombinant progeny from experimental crosses and Core A will isolate recombinant progeny from a further eight well conceived experimental genetic crosses between P. falciparum drug resistant and drug susceptible strains as part of this P01. The success of Core A will be aided by RP01 in efforts to maximize the number unique progeny from each cross. Additionally, RP01 will work closely with Core A to determine if bulk segregant analysis coupled with whole genome sequencing can speed the time taken to link genotype to phenotype. The phenotyping of progeny supplied by Core A and downstream mapping of genetic loci responsible for observed phenotypes are integral parts of RP01, RP02 & RP03 and such Core A is the linchpin of this P01. Successful creation of progeny for this P01 will further our understanding of the spread of artemisinin drug resistance and the emergence of piperaquine drug resistance.

抽象的 绘制引起耐药性表型的基因型的具体方法是进行经过深思熟虑 耐药性和药物易感性恶性疟原虫菌株之间的实验遗传交叉， 分离重组后代，然后使用定量性状局部映射将基因型与表型联系起来。三 恶性疟原虫遗传十字在脾切除的黑猩猩中进行了23年，其中之一是 这些十字的伟大成就是氯喹耐药性的遗传决定因素 - 中点突变 PFCRT基因。 NIH现在出于财务和道德原因禁止黑猩猩研究 开发了人肝小鼠模型（FRG Huhep小鼠），以取代黑猩猩 恶性疟原虫遗传杂交产生重组后代。 FRG小鼠缺乏Fumayl乙酰乙酸水解酶基因（F名称），这会导致肝细胞细胞 死亡。然而，肝细胞死亡由药物硝化酮控制。由于只有鼠标肝细胞缺乏 Fumayl乙酸乙酸酯水解酶，随着时间的流逝，这可以用人肝细胞对小鼠进行重生 闭合药物用于控制小鼠肝细胞死亡及其用人肝细胞替代的药物。在 与创建FRG Huhep鼠标的Yecuris Corporation密切合作，我们确保老鼠 我们将研究用于研究，具有最大的人类肝细胞嵌合体，并且容易受到恶性疟原虫的影响。 孢子岩感染。此外，小鼠能够在 HURBC输注及其允许小鼠中的恶性疟原虫肝脏阶段到血阶段过渡。下列的 去除血液，无性疟原虫血液阶段的体外扩张允许下游克隆和 然后 - 组分析和表型分析重组后代。 我们已经证明了我们使用FRG Huhep/hurbc鼠标的能力 来自实验十字和核心A的重组后代将与进一步的重组后代分离 恶性疟原虫耐药性和易感药物之间的八个构思良好的实验性遗传杂交 菌株作为此P01的一部分。 RP01将帮助核心A的成功以最大化数字 每个十字架的独特后代。此外，RP01将与核心A紧密合作，以确定是否散装分离 分析与整个基因组测序结合可以加快将基因型与表型联系起来所花费的时间。 由核心A提供的后代的表型和负责观察到的遗传位置的下游图 表型是RP01，RP02和RP03的组成部分，而此类核心A是该P01的关键。成功的 为此p01创造进步将进一步了解我们对青蒿素耐药性和 果皮耐药性的出现。