Experimental genetic crosses for malaria research

摘要 绘制导致耐药表型的基因图谱的具体方法是进行深思熟虑 恶性疟原虫耐药株和敏感株之间的实验遗传杂交， 分离重组子代，利用数量性状基因座定位，将基因与表型连锁。三 恶性疟原虫的遗传杂交是在切除脾的黑猩猩身上进行的，超过23年，其中一只 这些杂交组合的巨大成就是氯喹抗性点突变的遗传决定因素。 Pfcrt基因。出于经济和道德方面的原因，美国国立卫生研究院现在禁止黑猩猩的研究，但我们已经 开发了一种人-肝嵌合小鼠模型(FRG huHep小鼠)，以取代黑猩猩 恶性疟原虫遗传杂交重组后代的产生。 FRG小鼠缺乏富马酸乙酰乙酸酯水解酶基因(F代号)，这导致肝细胞 死亡。然而，肝细胞死亡是通过药物尼替松来控制的。因为只有小鼠肝细胞缺乏 富马酸乙酰乙酸酯水解酶，这使得随着时间的推移，小鼠能够与人肝细胞重新繁殖 用开关药物控制小鼠肝细胞的死亡及其与人肝细胞的替代。在……里面 与创造FRG huHep鼠标的Yecuris公司密切合作，我们确保MICE 我们在研究中使用了最大的人类肝细胞嵌合体，并且对恶性疟原虫易感。 子孢子感染。此外，小鼠能够保持人类红细胞(HuRBC)的数量 人红细胞输注，这允许恶性疟原虫在小鼠的肝期到血期的转变。跟随 血液去除，无性恶性疟原虫血液阶段的体外扩增允许下游克隆和 然后对重组后代进行组学分析和表型分析。 我们已经展示了我们有能力使用FRG huHep/huRBC小鼠来生成 来自实验杂交和核心A的重组后代将从进一步的 恶性疟原虫耐药和敏感的8个实验遗传杂交组合 菌株作为本P01的一部分。核心A的成功将得到RP01的帮助，努力使数字最大化 每个杂交组合都有独特的后代。此外，RP01将与核心A密切合作，以确定是否存在批量分离 分析与全基因组测序相结合可以加快将基因型与表型联系起来所需的时间。这个 A芯供体后代的表型鉴定及下游定位 表型是RP01、RP02和RP03的组成部分，而核心A是这一P01的关键。成功 P01子代的建立将进一步加深我们对青蒿素耐药性传播和 哌喹耐药性的出现。

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.