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Publication Date

2024-5

First Advisor

Steven A. Williams

Document Type

Honors Project

Degree Name

Bachelor of Arts

Department

Biochemistry

Keywords

biology, biochemistry, parasitology, genetics, neglected tropical diseases, nematodes, reverse genetic technology, filariasis

Abstract

Lymphatic filariasis is a neglected tropical disease that affects millions of people in countries throughout the tropics of Asia, Africa, and the Western Pacific. Caused by Brugia malayi, a mosquito-borne parasitic nematode that enters the human body upon a mosquito’s blood meal, lymphatic filariasis could lead to permanent morbidity with blockage of lymph drainage by the parasites. Current treatment strategies for lymphatic filariasis have limited effects, logistical challenges, and risks of drug resistance. The present study focuses on the biology of B. malayi at the L3 infective stage using reverse genetic techniques. Due to the irreproducibility of past transfection techniques on L3 B. malayi, we aimed to transfect the nematodes using an approach combining electroporation and lipofection to facilitate the delivery of genetic materials through the resistant cuticles of the nematodes. The expression of the transgene, green fluorescence protein (GFP), was assessed using fluorescence microscopy. The transcription of the transgene was further examined using reverse-transcription polymerase chain reaction (RT-PCR) on RNA isolated from transfected B. malayi. Both the fluorescence microscopy results and RT-PCR showed evidence of transient transfection. Green fluorescence was observed in body wall skeletal muscles, corresponding to where the plasmid promoter gene is expressed. RT-PCR results confirm intron splicing of GFP sequences in the transcript. Overall, we demonstrated that transfection of L3 B. malayi is possible with electroporation and lipofection.

Rights

©2024 Shilei Li. Access limited to the Smith College community and other researchers while on campus. Smith College community members also may access from off-campus using a Smith College log-in. Other off-campus researchers may request a copy through Interlibrary Loan for personal use.

Language

English

Comments

62 pages: color illustrations. Includes bibliographical references (pages 51-58).

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