Optimizing Luciferase Reporter Vector Transfection in 5L Rat Hepatoma Cells

Emily Calton, North Idaho College
Reilly Clark, Department of Biological Sciences, Boise State University
Ricky Aguayo, College of Southern Idaho
Jonathan Walsh, College of Southern Idaho
Cheri Lamb, Department of Biological Sciences, Boise State University
Ken Cornell, Department of Chemistry, Boise State University
Henry Charlier, Department of Chemistry, Boise State University
Kristen Mitchell, Department of Biological Sciences, Boise State University

Abstract

Signal transducer and activator of transcription (STAT) 1 is a transcription factor activated by the Jak/STAT pathway. When activated, STAT1 dimerizes and translocates to the nucleus, where it regulates transcription of target genes (Figure 1).¹ The major cytokine that induces the activation of STAT1 is interferon-gamma (IFNγ). In the presence of IFNγ, STAT1 forms a homodimer and binds to the interferon-gamma activated sequence (GAS). Binding of STAT1 to its response elements can be measured by luciferase reporting vectors. Cells transfected with these vectors will express luciferase, a firefly gene, in response to STAT1 binding to its response elements. The goal of this project was to optimize the transfection of 5L rat hepatoma cells with GAS and ISRE luciferase reporter vectors for the quantification of STAT1 response element activation following IFNγ treatment.

1. Leonard W. (2001) Role of Jakkinasesand STATs in cytokine signal transduction. International Journal of Hematology. 73:271-277