Combining Liver Stage Culture System with Backcross Genetics to Discover Antimalarial Drug Resistance Loci

项目摘要/摘要 在理解抗疟疾耐药性的分子基础方面取得了重大进展，主要是通过 利用遗传学的力量。由于有性结合发生在蚊子媒介中，克隆亲本的配子体 具有不同遗传背景和抗药性表型的品系可以混合，喂给蚊子，并且 在红细胞中产生的单倍体后代可以被克隆并用于识别与所需基因相关的遗传位点 表型。由于使用非人类的限制，与恶性疟原虫的遗传杂交受到了限制。 灵长类动物和/或植入人类肝细胞的小鼠的成本。在这里，我们提出了一种新的方法，使用 用于遗传杂交的肝期恶性疟原虫的高效、简便的体外培养体系。 回交育种是一种正向遗传学工具，可用于从一个品系中引入特定的遗传性状 排成第二行。大多数情况下，这在农业中被用来引入所需的特征(例如，抗病) 成为一条精英繁殖线。在此，我们提出了首次与恶性疟原虫进行表型辅助回交来鉴定 赋予或增强K13介导的青蒿素抗性的遗传位点。在目标1中，我们将进行回交 携带K13的恶性疟原虫药物敏感株(Nf54)和青蒿素抗性克隆的实验 E252Q突变和环期存活试验(RSA)中的耐药表型。亲本系的配子体 将混合，喂养蚊子和子孢子，用于接种384个培养良好的人类肝细胞。F1子代 将被收集并在改良的RSA中进行药物选择，以丰富抗药性后代，然后 与药敏亲本(Nf54)回交。我们将重复这个表型增强的回交三次 从回交4代(BC4)获得更多的倍数和克隆后代进行深度测序。在目标2中，我们将进行 克隆的BC4后代的表型和基因分析。青蒿素耐药表型将通过使用 BC4克隆后代的平板环期存活试验(RSA)。将进行QTL分析以确定基因座 将评估与青蒿素耐药性有关的K13 E252Q突变的作用。这项研究的结果 可以大大丰富人类疟疾寄生虫实验遗传学的工具箱，并揭示必要的 背景与青蒿素耐药性相关的E252Q K13突变。

PROJECT SUMMARY/ABSTRACT Major advances in understanding the molecular basis of antimalarial drug resistance have been achieved by using the power of genetics. Since sexual combination occurs in the mosquito vector, gametocytes of cloned parent lines with different genetic backgrounds and drug resistance phenotypes can be mixed, fed to mosquitos, and the resulting haploid progeny in erythrocytes can be cloned and used to identify genetic loci associated with the desired phenotype. Genetic crosses with Plasmodium falciparum have been limited due to limitations of using non-human primates and or costs of mice engrafted with human hepatocytes. Herein we propose a novel approach of using a highly efficient, facile in vitro culture system to produce liver stage P. falciparum for genetic crosses. Backcross breeding is a forward genetics tool that can be used to introduce a specific genetic trait from one line into a second line. Most often this has been used in agriculture to introduce a desired trait (e.g., disease resistance) into an elite breeding line. Herein we propose the first phenotype-assisted backcross with P. falciparum to identify genetic loci that either confer or enhance K13 mediated resistance to artemisinin. In Aim 1 we will conduct backcross experiments with drug susceptible (Nf54) and an artemisinin-resistant clone of P. falciparum that possesses K13 E252Q mutation and a resistant phenotype in the ring stage survival assay (RSA). Gametocytes of the parent lines will be mixed, fed to mosquitos, and sporozoites used to inoculate 384 well cultures of human hepatocytes. F1 progeny will be collected and subjected to drug selection in a modified RSA to enrich for resistant progeny and then backcrossed with the drug susceptible parent line (Nf54). We will repeat this phenotype-enhanced backcross three more times and clone progeny from backcross generation 4 (BC4) for deep sequencing. In Aim 2 we will conduct phenotype and genotype analysis of cloned BC4 progeny. Artemisinin resistance phenotypes will be assessed by using a plate-based ring stage survival assay (RSA) in cloned progeny from BC4. QTL analysis will be performed to identify loci associated with artemisinin resistance and the role of K13 E252Q mutation will be assessed. The results from this study could significantly enrich the toolbox for experimental genetics of the human malaria parasite and uncover essential background mutations that either confer or enhance E252Q K13 mutations in artemisinin resistance.