A Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-Infected Cells

In This Article

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 virus per 1 mL medium.
After desired h post-infection (hpi) (in this experiment at 10-12 hpi), transfect mRNA (500 ng of total mRNA per well o....

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Results

DNA transcription diagram with luciferase reporters; gel for mRNA analysis; RNA transfection setup.
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 R.......