Development of Tools for Stable Transfection in the Human Filarial Parasite Brugia malayi via the piggyBac transposon system

• Main Author: Chabanon, Johan
• Format: Others
• Published: Scholar Commons 2017
• Subjects: Lymphatic filariasis; Transfection; Molecular Biology; Parasitology; Public Health
• Online Access: http://scholarcommons.usf.edu/etd/6689; http://scholarcommons.usf.edu/cgi/viewcontent.cgi?article=7886&context=etd

Brugia malayi is one of three species of nematode known to cause lymphatic filariasis (LF) in humans. LF infects over 120 million people, causing debilitating disease. Various global programs have been launched in the past 20 years to eliminate LF. These programs have greatly scaled up the resources and efforts allocated to halting the transmission and reducing disease burden. Only a few drugs are used to treat LF, and resistance is thus a devastating possibility. Research aimed at identifying new drug targets could therefore prove essential in elimination of LF. Genetic manipulation of B. malayi has been limited to transient transfections. A transfection system allowing for stable integration of transgenic sequences into the nuclear genome of this parasite would enable more robust studies that could lead to identification of novel drug targets and vaccine candidates. The piggyBac (pB) transposon system has been successfully applied to develop a stable transfection system in a variety of species. This system involves two plasmids, a helper and a donor. The donor plasmid contains the target DNA and a selectable marker flanked by specific inverted terminal repeat (ITR) regions. The helper plasmid expresses the pB transposase that will catalyze the precise integration of any DNA report tools necessary to adapt the pB system in B. malayi. Three versions of the donor plasmid were constructed, each containing a Gaussia Luciferase (GLuc) selectable marker but differing only by the fluorescent protein expressed. The construct containing a YFP gene was used to transfect embryos via biolistics to test whether YFP and GLuc are expressed.