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Publication Date

2022-05-09

First Advisor

Steven A. Williams

Document Type

Honors Project

Degree Name

Bachelor of Arts

Department

Biochemistry

Keywords

CRISPR, genome editing, neglected tropical diseases, brugia malayi, lymphatic filariasis, mosquito, pathogen, parasite, lymphatic system, elephantiasis, genomics, polymerase chain reaction, parasitic diseases, transfection, nucleases, vector cloning

Abstract

There is an ongoing effort to eradicate lymphatic filariasis (LF), a Neglected Tropical Disease affecting over 120 million people around the world, caused by mosquito-borne filarial parasites. The filariae live within the human lymphatic system and block lymph drainage, leading to lymphodema (tissue swelling) or elephantiasis (tissue thickening). There are several effective drugs against LF, but they are limited in supply, must be given repeatedly over long periods, and require a logistically difficult and expensive distribution process. Reverse genetic techniques are an essential tool for developing new treatments and recognizing drug resistance pathways. Here, we report our data using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in order to introduce Gluc and GFP reporter genes via homology-directed repair into Brugia malayi . L3 stage B. malayi were successfully co-cultured in vitro with BESM cells and lipofected over 8-day periods. Luminescence and fluorescence assays to confirm expression of transgenes were identified as areas in need of further improvement, as well as nested PCR protocols for DNA examination of plasmid insert. Once optimized, this method will be used to report genes thought to be involved in the development of drug resistance, as well as to experimentally knock-out genes involved in biochemical pathways to determine new drug targets.

Rights

©2022 Sophia Chertock. 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

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