Summer Research Program for Science Teachers

Michael Barr

Scarsdale High School, Westchester



Assessment of protein function through the use of gene knockouts in mammalian cell culture


Objective:  Students will determine the function of a cellular protein by designing an experiment that compares a “knockout” cell line with a “wildtype” cell line. STANDARD A

Background:  As a prerequisite for this lab students should have a good understanding of cell functions and genetics.  They need to know what a knockout is and how it can be used to help learn the function of a given protein.

Materials:  Pipettes (micro and power), Petri Dishes or other cell culture flasks, Phase contrast Microscopes, Media, Trypsin, PBS and other cell culture chemicals. An Incubator (with CO2), Refrigerator, and a Hood would all be nice.  Other materials will be needed depending on what experiments are designed.  The cells will have to be ordered from biotech companies or academic laboratories.

Motivation: Students will get a chance to do real science experiments and possibly make novel findings that can contribute to future research.

Procedure:  There is not an exact procedure for this lab.  There will be many different possibilities depending on what experiments the students design and what protein you choose to look at.  What cells are used really depends on what equipment and facilities your schools has.  Mammalian cells will require 37 °C and 5% CO2 incubators.  Students will have to spend the first month or so learning the basics of cell culture and how to maintain cells.  They must learn aseptic technique and how to design experiments.

Below is one possible procedure to determine the function, if any, Myosin II has in cell motility.


Does Myosin II play a crucial role in cell motility?

Obtain Myosin II -/- (knockout)  and Myosin II +/+ (control) cell lines (Fibroblasts or 3T3s).  Grow confluent monolayers of each cell line in separate Petri dishes (or six wells etc.).  One way to measure cell motility is will a simple “wound healing assay.”  Using a 200 microliter pipette tip scrape a line down the center of the cells in the Petri dish.  Over the next 24 hours monitor the speed of wound closure.  If myosin II is crucial in cell motility you would not expect any wound closure in your experimental.  If myosin plays no role, there should be no difference in data from experimental to control.

In a second part of this experiment a cell line with myosin II transfected back into the cells should be obtained and the experiment should be repeated with this line.  Myosin is a good protein to use because there are many classes of  it and it has many different and important functions in organisms and cells, ranging from inflammatory response to metastasis in cancer.


Return to Biology Lesson Plan Menu