EP_054. Enhanced lentiviral GFP expression driven by CMV promoter: Towards successful generation of induced neural progenitor cells (iNPCs) through direct reprogramming

Nur Jannaim Muhamad^1,2*, Fatimah Diana Amin Nordin¹, Rosnani Mohamed¹, Mohd Khairul Nizam Mohd Khalid¹, Julaina Abdul Jalil¹, Nurul Husna Abd Razak², Muhammad Fauzi Daud², Izyan Mohd Idris¹

1. Inborn Errors of Metabolism & Genetics Unit, Nutrition, Metabolism & Cardiovascular Research Centre, Institute for Medical Research, National Institutes of Health, Ministry of Health, Malaysia.

2. Institute of Medical Science Technology, Universiti Kuala Lumpur, Malaysia

*Corresponding author: Nur Jannaim Binti Muhamad. This email address is being protected from spambots. You need JavaScript enabled to view it.

ABSTRACT

INTRODUCTION: Generating induced Neural Progenitor Cells (iNPCs) through direct reprogramming depends on high and robust overexpression of transcription factors. Lentiviral vectors are widely used as a delivery tool for transcription factors due to their ability to support efficient expression. Promoter selection plays a key role in determining expression levels and the overall success of direct reprogramming into iNPCs.

OBJECTIVE(S): This study aimed to compare the efficiency of two lentiviral promoters which are cytomegalovirus (CMV) promoter and elongation factor-1 alpha promoter (EF1a) promoter) in driving green fluorescent protein (GFP) expression in lentiviral systems using Lenti-X 293T cells.

MATERIALS & METHODS: Lentiviral expression plasmids were constructed by cloning the GFP reporter gene alongside either the CMV or EF1α promoter into lentiviral vector backbone. Lenti-X 293T cells were transfected using fourth-generation lentiviral packaging systems.  GFP expression was assessed 72 hours post-transfection via flow cytometry. Fluorescence microscopy was used for visual confirmation. Statistical analysis was performed using unpaired t-tests with p < 0.05 considered significant. 

RESULTS: The comparison of both promoters revealed enhancement of CMV-driven GFP expression during lentiviral transfection. A significantly higher percentage of GFP expressing cells was observed in cells transfected with CMV-driven lentiviruses (mean: 99.13333, SEM: ±0.55478) compared to EF1α-driven lentiviruses (mean: 52.4, SEM: ±0.60828). The difference in mean GFP expression was highly significant (p < 0.001). Fluorescence microscopy confirmed more intense and widespread GFP fluorescence in cells transfected with lentiviral constructs containing the CMV promoter.

CONCLUSION: These findings indicated that the CMV promoter is more efficient at driving GFP expression in lentiviral systems, highlighting its suitability for achieving high and consistent overexpression of transcription factors essential for successful iNPC reprogramming.

KEYWORDS: Direct Reprogramming, induced Neural Progenitor Cells (iNPCs), Green Florescence Protein (GFP), Promoter, Lentivirus Production