Question

For questions 5 through 9: You have isolated an epithelial cell line which is unresponsive to EGF (normal epithelial derived cell lines require EGF in the culture media for growth). You suspect that EGF receptor signaling in this cell line is impaired, and you designate this a mutant cell line. As an assay for EGFR signaling, you have stably transfected this mutant cell line and a normal cell line with a luciferase-based reporter construct that is responsive to the transcription factor Elk-1. You want to determine what component of the signaling pathway is impaired. 5. (5 pts) Briefly describe a method you could use to transfect the luciferasebased reporter construct DNA into a cell, and then describe how you could establish (or select for) a stable cell line expressing this reporter construct.

Answer 1

In order to create stable cell line which expresses luciferase construct, we need to clone the luciferase gene in the pCDNA3.1 plasmid. This plasmid has neomycin antibiotic resistance gene.

Before transfecting the mutant and wild-type cells we need to make the kill curve of the cell lines with neomycin resistance. We need to select antibiotic concentration as whcih90% of the cells shows cell death.

Once we transfected the cell with pCDNA3.1 we need to select the transfected cells with neomycin antibiotic. Cells which have pcDNA plasmid will be able to survive in the presence of neomycin whereas cell which doesn't have transferred plasmid will be killed.

Once we have only very few cells in the dishes we need to split the cells in such a way that each well of 96 well plate has 1-2 cells.

We need to grow cell till they from the colony. Once we have enough cells we need to check whether cells are expressing luciferase or not. We can check expression either by western blot using anti-luciferase antibody or by real-time PCR