A Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-Infected Cells
Published: February 29, 2024
Abstract
Source: Dhungel, P., et al. In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells. J. Vis. Exp. (2019)
This video demonstrates an assay to study the translation regulation in poxvirus-infected cells. Uninfected and virus-infected cells were co-transfected with mRNAs encoding for firefly luciferase (Fluc) and Renilla luciferase (Rluc), with the Fluc mRNA containing a 5' Poly(A) leader sequence. Upon adding bioluminescent substrates on lysed cells, measure the signal from both the luciferases to identify the translational advantage of the Fluc mRNA in virus-infected cells, conferred by the leader sequence.
Protocol
1. Transfect mRNA to Cells Seed HeLa cells in a 24-well plate (to be approx. ~80-90% confluent the next day) and incubate overnight in an incubator at 37 °C with 5% CO2. Infect HeLa cells with vaccinia virus (VACV) at a Multiplicity of Infection (MOI) of 5 or keep uninfected HeLa cells for comparison. NOTE: MOI is the number of infectious viral particles per cell. MOI of X= {[(Number of cells * X) / Virus Titer] * 1000} µl of vi…
Representative Results
Figure 1: mRNA synthesis and transfection. (A) Schematic of in vitro transcription. DNA amplified by PCR containing the luciferase gene downstream from the 5'-UTR of interest and the T7 promoter is used as the template. The T7 RNA polymerase is recruited to the promoter and adds ribonucleotides, shown in white, from 5' to 3' direction. Once mRNA is 25-30 nt l…
Divulgazioni
The authors have nothing to disclose.
Materials
| Corning 96 Well Half-Area white flat bottom polystyrene microplate | Corning | 3693 | Opaque walled 96 well white plate with solid bottom |
| Dual-Luciferase Reporter Assay System | Promega | E1960 | Dual-Luciferase Assay Kit (DLAK) |
| GloMax Navigator Microplate Luminometer | Promega | GM2010 | Referred as multimode plate reader luminometer |
| Lipofectamine 2000 | Thermo Fisher Scientific | 11668019 | Cationic lipid transfection reagent |
| Opti-MEM | Thermo Fisher Scientific | 31985070 | Reduced serum media |
Tags
Citazione di questo articolo
A Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-Infected Cells. J. Vis. Exp. (Pending Publication), e22002, doi: (2024).