To access this work you must either be on the Smith College campus OR have valid Smith login credentials.

On Campus users: To access this work if you are on campus please Select the Download button.

Off Campus users: To access this work from off campus, please select the Off-Campus button and enter your Smith username and password when prompted.

Non-Smith users: You may request this item through Interlibrary Loan at your own library.

Publication Date

2025-5

First Advisor

Steven A. Williams

Document Type

Honors Project

Degree Name

Bachelor of Arts

Department

Biochemistry

Keywords

CRISPR, infectious disease, mosquitoes, immunity

Abstract

The Aedes genus of mosquitoes are significant vectors for multiple neglected tropical diseases (NTDs) such as dengue fever, yellow fever, and lymphatic filariasis. Aedes albopictus is specifically native to the tropical and subtropical areas of Southeast Asia and is considered to be a potential vector for various bacterial, viral, and microfilarial transmission. Editing genes responsible for mosquito immunity potentially prevent the spread of these diseases by increasing immune activation in mosquitoes against the pathogens that cause the diseases. The Toll pathway plays an important role in mosquitoes' innate immune defense against fungal, bacterial, viral, and microfilaria infections. Degradation of Cactus, a negative regulator located in the Toll pathway, upregulates gene expression for antimicrobial peptides (AMPs) such as defensin and cecropin, which are the predominant and immune-inducible AMPs in mosquitoes. We hypothesize that CRISPR-mediated knock-out of Cactus gene could yield an upregulation of immune response. The study aims to transfect the C6/36 mosquito cell line with a pAc-sgRNA-Cas9 plasmid and a knock-in puromycin selection cassette. Successfully transfected cells are selected in puromycin medium. CRISPR on-target and off-target regions are assessed by PCR and Sanger sequencing. Expression differences of immune genes in Cactus knock-out cells are detected by RT-qPCR. Relative expression level, calculated in 2^-ΔΔCT, is analyzed using one-way ANOVA among clones for each gene of interest.

Rights

©2025 Wanying Fan. 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

80 pages: color illustrations, charts. Includes bibliographical references (pages 76-80).

Download

Smith Only:
Off Campus Download

Share

COinS