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oai:keep.lib.asu.edu:node-2008682025-06-02T18:38:20Zoai_pmh:alloai_pmh:repo_items

200868 https://hdl.handle.net/2286/R.2.N.200868 http://rightsstatements.org/vocab/InC/1.0/ http://creativecommons.org/licenses/by-nc-sa/4.0 2025-05 30 pages Lariego, Ava Claire Bartelle, Benjamin Borges Florsheim, Esther Ochoa Zermeño, Santiago Barrett, The Honors College Harrington Bioengineering Program School of Biological & Health Systems Engineering Efficient transfection is essential for genetic engineering and biological research applications, yet optimizing protocol for different payloads and cell types remains challenging. Untranslated regions (UTRs) are non-coding sequences derived from Zika virus (ZIKV) that influence RNA stability and translation efficiency. This study evaluated linear DNA as a rapid, PCR-based alternative to plasmid DNA for transient gene delivery, particularly in the context of high-throughput experiments. The goal was to determine whether incorporating UTRs could enhance expression stability in linearly transfected cells. Human embryonic kidney (HEK293) and human microglial (HMC3) cells were transfected with GFP constructs delivered via plasmid, linear, or viral transfection methods, both with and without ZIKV UTRs. GFP expression was tracked over 10 days, and fluorescence decay rates were analyzed using linear regression. Linear transfection was validated as a viable technique in both cell lines, demonstrating comparable expression behavior to plasmid and viral methods. However, the addition of ZIKV UTRs did not significantly alter GFP decay rates in any method. These findings suggest that while linear DNA offers advantages in speed and scalability, UTRs alone may be insufficient to sustain expression. Future work should explore additional viral elements or delivery enhancements to improve expression profiles in transfection systems. Linear transfection Zika virus (ZIKV) Untranslated regions (UTRs) GFP expression RNA stability HEK293 cells HMC3 microglia Flow Cytometry Enhancing RNA Stabilization through Flavivirus Genome Cyclization: A Comparative Assessment of Transfection Methods