Experimental genetic crosses for malaria research

摘要 绘制导致耐药表型的基因型的一种具体方法是进行一个经过深思熟虑的 抗药性和药物敏感性恶性疟原虫株之间的实验性遗传杂交， 分离重组后代，然后使用数量性状基因座作图将基因型与表型联系起来。三 P.恶性疟原虫基因杂交在切除脾脏的黑猩猩身上进行了23年，其中一个 这些杂交的巨大成就是氯喹抗性的遗传决定因素-- pfcrt基因美国国立卫生研究院现在已经禁止黑猩猩研究的经济和道德的原因，但我们有 开发了一种人肝嵌合小鼠模型（FRG huHep小鼠），以取代黑猩猩， 从恶性疟原虫遗传杂交产生重组后代。 FRG小鼠缺乏富马酰乙酰乙酸水解酶基因（F命名），这导致肝细胞 死亡然而，肝细胞死亡是控制与药物nitisinone。因为只有小鼠肝细胞缺乏 富马酰乙酰乙酸水解酶，这使得随着时间的推移，小鼠能够用人肝细胞重新增殖， 开-关药物使用以控制小鼠肝细胞的死亡及其用人肝细胞的替代。在 与Yecuris公司密切合作，谁创造了FRG huHep小鼠，我们确保小鼠 我们用于我们研究的人肝细胞嵌合体最大，对恶性疟原虫易感 子孢子感染此外，小鼠能够在免疫后维持人红细胞（huRBC）群体。 huRBC输注，这允许小鼠中恶性疟原虫肝脏阶段到血液阶段的转变。以下 血液去除后，无性恶性疟原虫血液阶段的体外扩增允许下游克隆， 然后对重组后代进行组学分析和表型分析。 我们已经证明了我们使用FRG huHep/huRBC小鼠产生 核心A将从实验杂交中分离重组后代，核心A将从另一个杂交中分离重组后代。 八个构思良好的恶性疟原虫耐药和敏感的实验性遗传杂交 作为P01的一部分。核心A的成功将得到RP 01的帮助，以最大限度地提高 每一次杂交都有独特的后代此外，RP 01将与岩心A密切合作，以确定是否存在大量偏析物。 结合全基因组测序的分析可以加快将基因型与表型联系起来的时间。的 由核心A提供的后代的表型和负责观察到的遗传位点的下游作图 表型是RP 01、RP 02和RP 03的组成部分，而核心A是P01的关键。成功 这种P01的后代的产生将进一步加深我们对青蒿素耐药性传播的理解， 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.