More on Experimental Design
Why did you decide to do this experiment?
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In this lab, you are a graduate student or post-doctoral fellow in a
circadian biology research laboratory. Your research group has decided
to focus the studies of the lab on the period gene. In the past, your
colleagues collected hundreds of flies to extract mRNA to measure period
gene mRNA changes during daily, or circadian, cycles. You figure there
must be a better way.
Why not find a way to measure period gene changes in an individual fly?
But how? All genes contain a regulatory region, the promoter, that lies
"upstream," or before the coding region of the gene. When the
appropriate signals turn on the promoter, the DNA is transcribed into
mRNA. Perhaps there is a way to quantify whenever this switch is turned
on, and thus have a measure of the timing of when the period gene is
turned on. It turns out there are a number of clever ways to do just
Reporter genes do what their name implies: they "report" on the
activity of the promoter to which they are linked. Reporter genes come
from many different types of organisms. The key ingredient is that
the reporter have some sort of novel appearance or easy way to be
measured. Green fluorescent protein from jellyfish and luciferase from
fireflies are some of the most popular reporter genes. Because you are
looking at circadian rhythms that fluctuate over the period of a day,
you decide you need an enzyme reporter gene that has a short half-life.
Luciferase is the enzyme that converts the chemical luciferin into light.
In the presence of luciferin, luciferase has a half-life of 2-4 hours,
short enough to show fluctuations within a day. You decide that
luciferase is the best reporter gene for your system.
Won't it be interesting to turn an ordinary fruit fly into a fly that
glows? Your fruit fly will be very different from fireflies. Your
fruit fly will be a new organism genetically engineered in a
laboratory. Unlike fireflies that have evolved light-producing
mechanisms to attract mates, your glowing fly will produce light that,
in most circumstances, will be undetectable to the eye. Special
machines will detect the low levels of light produced. Further, the
light produced has no practical function for your fruit fly. Rather,
the light produced is your window into the molecular clock in the fly
that regulates its daily rhythms. (The experiments in this lab are based
on actual experiments [see Brandes et al. 1996].)
Now that you have thought through the technical strategies to create the
glowing fly, examine your hypothesis for the experiment:
Transgenic per-luc flies will produce light in a pattern that reflects
the transcriptional activity of the period gene.