Upon completion of this module topic, you should:
- Be able to analyze gene expression resulting from a cell transfection experiment, using luciferase or GFP reporter constructs.
- Be able to prepare cell lysates from transfected cells by suspending adherent cells and lysing cells.
- Be able to conduct a luciferase assay on cell lysates by carrying out luciferase reactions and using bioluminescence to measure luciferase expression.
- Be able to use a luminometer to measure bioluminescence of cell lysate samples.
Preparation of Cell Lysates
This is Part A, Preparation of Cell Lysates, under the module topic, Cell Culture Techniques. This topic part has two sections: Content Tutorials & Animation.
1. MIT Module (2.2): Transfection When you access the MIT website, refer to all of the information provided on the webpage including the Introduction, and the Protocols Part 1 and 2 listed below.
Introduction & Protocols: Luciferase Assay Overview
Part 1: Transfection
Part 2: Practice Luciferase Reactions
- Overview (ocw.mit.edu, HTML Page)
2. MIT Module (2.3): Luciferase Assays and RNA Prep When you access the webpage, refer to the information provided in the following sections: Protocols: Part 1: Preparing Cell Lysates Part 2: Luciferase Assays
- Luciferase Assays (ocw.mit.edu, HTML Document)
3. MIT – GFP Reporter Construct, Cell Lysis & Evaluating GFP expression
Lipofection Information (Transfection & GFP Reporter Construct) When you visit the web page you will read an introduction that describes the process of cell transfection with a focus on one type of reporter construct that encodes for Green Fluorescent Protein (GFP).
- Lipofection Information (ocw.mit.edu, PDF Document)
4. Transfection Reference Guide – Promega When you access the reference guide, refer to Chapter 6: Genetic Reporter Systems. You will learn about several types of common genetic reporter systems used to study eukaryotic gene expression such as the firefly luciferase, renilla luciferase, Green Fluorescent Protein (GFP), and Beta Galactosidase reporter systems to name a few.
- Transfection Reference Guide (www.promega.com, PDF Document)
The following web link will take you to the Science Launcher website where you will view an animation about reporter constructs that will briefly depict how they function and are expressed in a cell. Reporter constructs are used many times in cell culture transfection experiments because they provide a way to visualize and test for the expression of a particular gene that is located within the plasmid(s) that you have transfected into cells.
- Science Launcher Website (sciencelauncher.com, Multimedia Page)