Institute of Biomedical Sciences

Title

New genetic tools to study gene function in fatal African sleeping sickness parasite

Poster Number

4

Document Type

Poster

Status

Graduate Student - Doctoral

Abstract Category

Basic Biomedical Sciences

Keywords

parasite, forward-genetics, gain-of-function, Trypanosoma

Publication Date

Spring 2018

Abstract

African trypanosomes are unicellular parasites that result in death if left untreated. Treatment of African trypanosomiasis relies on six effective drug treatments, which can be highly toxic and difficult to administer. While there are on-going studies to develop new drugs, resistance to existing drugs is a persistent threat. Trypanosomes are an early branching eukaryote with only limited homology to highly studied genomes; more than 60% of the Trypanosoma brucei genome remains annotated as hypothetical genes of unknown function. To better understand basic biology, pathogenesis, and resistance of this parasite, we need to uncover gene functions and regulations.

To accomplish this goal, we have developed a whole-genome gain-of-function library to apply in forward genetic screens and identify genes associated with a wide range of phenotypes. Gene expression regulation in Trypanosomes required that we build an ORF-based (PCR amplified) library, which was cloned into a Gateway library for introduction into the Trypanosoma brucei genome by transfection. Preliminary genetic screens demonstrated that we were able to successfully incorporate 90% of the intended gene products into the genome with greater than 10-fold coverage. However, we encountered two challenges: 1) individual transfections result in different sets of genes represented and 2) inconsistent gene expression among transfections.

Here, we have implemented steps for the overall normalization and consistent usage of our cloned overexpression library. Primarily, we undertook a process to produce a cell line that will permit the consistent, highly inducible expression of our library among multiple transfections. In addition, we have streamlined our transfection method to focus on producing a consistent population of cells that all harbor the same subset of library genes with the desired level of coverage. To further normalize the variance, we made our library cell line and expanded it to create a large bank of library cells for future genetic screens. With these steps now completed, we have prepared next-generation sequencing libraries to fully assess the expression and consistency. With this forward genetic tool in hand, we will be able to conduct genetic screens to uncover pathways of drug resistance, pathogenesis, and other key biological features of this devastating human parasite.

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New genetic tools to study gene function in fatal African sleeping sickness parasite

African trypanosomes are unicellular parasites that result in death if left untreated. Treatment of African trypanosomiasis relies on six effective drug treatments, which can be highly toxic and difficult to administer. While there are on-going studies to develop new drugs, resistance to existing drugs is a persistent threat. Trypanosomes are an early branching eukaryote with only limited homology to highly studied genomes; more than 60% of the Trypanosoma brucei genome remains annotated as hypothetical genes of unknown function. To better understand basic biology, pathogenesis, and resistance of this parasite, we need to uncover gene functions and regulations.

To accomplish this goal, we have developed a whole-genome gain-of-function library to apply in forward genetic screens and identify genes associated with a wide range of phenotypes. Gene expression regulation in Trypanosomes required that we build an ORF-based (PCR amplified) library, which was cloned into a Gateway library for introduction into the Trypanosoma brucei genome by transfection. Preliminary genetic screens demonstrated that we were able to successfully incorporate 90% of the intended gene products into the genome with greater than 10-fold coverage. However, we encountered two challenges: 1) individual transfections result in different sets of genes represented and 2) inconsistent gene expression among transfections.

Here, we have implemented steps for the overall normalization and consistent usage of our cloned overexpression library. Primarily, we undertook a process to produce a cell line that will permit the consistent, highly inducible expression of our library among multiple transfections. In addition, we have streamlined our transfection method to focus on producing a consistent population of cells that all harbor the same subset of library genes with the desired level of coverage. To further normalize the variance, we made our library cell line and expanded it to create a large bank of library cells for future genetic screens. With these steps now completed, we have prepared next-generation sequencing libraries to fully assess the expression and consistency. With this forward genetic tool in hand, we will be able to conduct genetic screens to uncover pathways of drug resistance, pathogenesis, and other key biological features of this devastating human parasite.