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

肝阶段培养系统与回交遗传学相结合发现抗疟药物耐药位点

• 批准号: 9891003
• 负责人: DENNIS E KYLE
• 金额: $ 18.88万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-11 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9891003
• 关键词: Address; Agriculture; Anti-malarial drug resistance; Aotus primate; Artemisinins; Backcrossings; Biological Assay; Biology; Breeding; Chloroquine resistance; Coculture Techniques; Combined Modality Therapy; Culicidae; Data; Disease Resistance; Drug resistance; Erythrocytes; Experimental Genetics; Generations; Genes; Genetic; Genetic Crosses; Genotype; Haploidy; Hepatocyte; Human; Human Genetics; In Vitro; Injections; Ivermectin; Laboratories; Liver; Logistics; Malaria; Mediating; Methods; Molecular; Morbidity - disease rate; Mus; Mutation; Pan Genus; Parasites; Parasitic nematode; Parents; Pharmaceutical Preparations; Phenotype; Plasmodium falciparum; Procedures; Public Health; Quantitative Trait Loci; Recombinants; Research; Resistance; Role; Sporozoites; System; Thailand; Time; Work; base; cost; deep sequencing; experimental study; forward genetics; genetic approach; genomic locus; global health; humanized mouse; in vitro Model; in vivo; mortality; mutant; nonhuman primate; novel; novel strategies; resistance gene; tool; trait; vector mosquito

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.

项目总结/摘要 在了解抗疟药物耐药性的分子基础方面取得了重大进展， 利用基因的力量由于性结合发生在蚊子载体中，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， 可以克隆得到的红细胞中的单倍体后代，并用于鉴定与所需的 表型与恶性疟原虫的遗传杂交由于使用非人类的限制而受到限制 灵长类动物和/或移植人肝细胞的小鼠的成本。在此，我们提出了一种新的方法， 高效、简便的体外培养系统，以产生用于遗传杂交的肝期恶性疟原虫。 回交育种是一种正向遗传学工具，可用于从一个品系引入特定的遗传性状 到第二行。这在农业中最常用于引入期望的性状（例如，抗病性） 变成了一个优良的繁殖系在此，我们提出了第一个表型辅助回交与恶性疟原虫，以确定 这些基因座赋予或增强K13介导的对青蒿素的抗性。在目标1中，我们将进行回交 用具有K13的恶性疟原虫的药物敏感（Nf 54）和青蒿素抗性克隆进行实验 E252 Q突变和环期存活试验（RSA）中的耐药表型。亲本系的配子体 将被混合，喂给蚊子，子孢子用于培养人肝细胞的384孔培养物。F1后代 将被收集并在改良的RSA中进行药物选择以富集抗性后代，然后 与药物敏感亲本系（Nf 54）回交。我们将重复这种表型增强回交三 更多次，并从回交4代（BC 4）克隆后代用于深度测序。在目标2中，我们将 表型和基因型分析克隆的BC 4后代。青蒿素抗性表型将通过使用 在来自BC 4的克隆后代中进行基于平板的环期存活测定（RSA）。将进行QTL分析以确定基因座 将评估与青蒿素耐药相关的基因突变和K13 E252 Q突变的作用。本研究的结果 可以大大丰富人类疟疾寄生虫实验遗传学的工具箱， 在青蒿素抗性中赋予或增强E252 Q K13突变的背景突变。