Summer
Research Program for Science Teachers
Scarsdale High School, Westchester
2003
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.