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Poster
Investigating the Effects of HER2 cDNA Transfection and Identifying Novel Binding Partners using Phage Display
University of West Florida Libraries
Summer Undergraduate Research Program (University of West Florida, Pensacola, Florida, 08/2023)
10/14/2023
Abstract
INTRODUCTION • Human Epidermal Growth Factor Receptor 2 (HER2 receptors, RTK), while naturally occurring in normal tissues at lower levels, are found to be overexpressed or amplified in cancers; such as breast, gastric, ovarian, lung, and colorectal. • This can increase cell signaling through the HER2 pathway, leading to aggressive and uncontrolled cancer cell growth and survival in the human body. • Currently, 20% of all diagnosed breast cancer in the U.S. are HER2 positive. (BCRF 3/22/23) • Targeting and blocking the HER2 receptors can inhibit cell growth, inducing cell death, and activate the immune system. • Phage display is a bacteriophage screening method that identifies phage capable of binding to specific receptors, such as HER2 RTKs. • Current (FDA approved) targeted HER2 therapies: Trastuzumab (Herceptin) and Pertuzumab (Perjeta). AIMS • Observe the optimal conditions for, and effects of, the transfection of HER2 cDNA with Human Embryonic Kidney cells (HeK293 cells). • Test the effects of HER2 agonists upon application to transfected cells • Identify bacteriophage with the unique ability to specifically bind to HER2 receptors. • Identify phage that inhibit HER2 activity. METHODS • HER2 cDNA was grown in E.coli, extracted, and purified using a DNA mini-prep kit. • The extracted cDNA was then confirmed through external DNA sequencing and gel electrophoresis. • Subsequently, it was replicated using a DNA maxi-prep kit before being transfected into HEK cells using calcium phosphate precipitant. • Once transfected, the cells were monitored and imaged via EVOS microscope. Florescence microscopy was utilized to show protein expression: HER2 tagged with Green Fluorescent Protein (GFP) and nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI). RESULTS • The HER2 plasmid was confirmed through addgene sequence comparison, gel electrophoresis, DNA concentration (1297.3 ng/uL per nanodrop data). • Transfection agent TurboFect performed poorly (cell clumping & detachment). Calcium phosphate reagent improved transfection results. • GFP present at 24 hours, intensity and the concentration of GFP-producing cells peaked at 48 hours, cells maintained GFP signals for up to five days, and passaged transfected cells maintained GFP signal. • HER2-specific phage libraries from the PHD7 and C7C libraries will be developed. uwf.edu/biology
Details
- Title
- Investigating the Effects of HER2 cDNA Transfection and Identifying Novel Binding Partners using Phage Display
- Resource Type
- Poster
- Event
- Summer Undergraduate Research Program (University of West Florida, Pensacola, Florida, 08/2023)
- Contributors
- Rodney P Guttmann (General Contributor) - University of West Florida, Hal Marcus College of Science and Engineering
- Publisher
- University of West Florida Libraries; Argo Scholar Commons
- Format
- pdf
- Copyright
- Permission granted to the University of West Florida Libraries by the author to digitize and/or display this information for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires the permission of the copyright holder.
- Identifiers
- 99380469397006600
- Academic Unit
- Biology; Hal Marcus College of Science and Engineering ; Summer Undergraduate Research Program 2023
- Language
- English