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

> **NIH NIH R21** · UNIVERSITY OF GEORGIA · 2020 · $188,750

## Abstract

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.

## Key facts

- **NIH application ID:** 9891003
- **Project number:** 5R21AI144591-02
- **Recipient organization:** UNIVERSITY OF GEORGIA
- **Principal Investigator:** DENNIS E KYLE
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $188,750
- **Award type:** 5
- **Project period:** 2019-03-11 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9891003

## Citation

> US National Institutes of Health, RePORTER application 9891003, Combining Liver Stage Culture System with Backcross Genetics to Discover Antimalarial Drug Resistance Loci (5R21AI144591-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9891003. Licensed CC0.

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