Hello everyone, I'm Florian Pontheaux, a 3rd-year PhD student in the team
and I will present you the practical four.
As you saw in the previous video, sea urchin early embryos are a powerful model to study
mRNA translation regulation in relation to cell cycle regulation.
The fertilisation will trigger the stimulation of protein synthesis to allow the first
mitotic division of the embryos. In our laboratory, we identified by polysomal
profiling which mRNA are specifically translated one hour following fertilisation.
In eukaryote cells, mature mRNA present basic structural elements that are required
to keep the mRNA functional, including a 5' cap, a 5' untranslated region,
an open reading frame, a 3' untranslated region, and a poly A tail structure.
Cis-regulating elements are often found in the UTRs of specific mRNA.
Therefore, it is interesting to study the role of specific UTRs in controlling the mRNA
translational activity. For this purpose, we will use our luciferase reporter mRNA
containing a 5' cap. The ORF will encode for a Firefly luciferase, and is placed in
between a 5' and a 3' UTR of Xenopus globin mRNA.
The Xenopus globin mRNA is known to present high efficiency for promoting the
translation of mRNA. Luciferase is a popular choice as a reporter because
the enzyme is functional immediately upon translation and the detection assay is rapid,
sensitive, and quantitative. Here we will show you how to introduce by microinjection
the construct into sea urchin eggs and how to detect translational activity controlled by
your specific UTR. Here you will learn how to prepare the eggs, and more specifically
how to gently remove the egg jelly.
Then you will see how to prepare a specific glassware set up and pipettes
as the experiment is designed to be performed in triplicate.
Next will be how to align the eggs and perform microinjection into the eggs,
and at last you will learn how to recover the eggs from the glassware setup
and monitor the Luciferase reporter's translational activity.
The obtained data will give you an idea of how you can study the role of the different
element structures of mRNA in translational regulation.
To begin, we prepare freshly spawned eggs by gently treating them with citric acid
to digest the egg jelly layer.
For that we use four agarose Petri dishes with a 1% agarose gel.
The first is filled with the citric acid solution and the three others with natural filtered seawater.
Then, to delly eggs, they are dropped in the citric acid solution for no more than
one minute. Otherwise, you will cook your eggs.
Eggs are swirled and concentrated in the centre of the Petri dish.
With the Pasteur pipette, the maximum of eggs in the minimum of solution are
transferred into the next agarose plate in order to wash them.
Repeating this process, they are successively transferred into the third and fourth plate.
Dejellied eggs can be stored at 17°C for up to three hours without fertilisation problems
but in our lab they are preferentially used in the hour following treatment.
Don't worry about losing eggs between washes. We still end up with thousands
of eggs, much more than needed.
To microinject eggs, we need to maintain them on a surface so they will not roll
away during microinjection.
To do this, we choose to stick them on coverslips.
So coverslips are soaked in a 1% protamine sulphate solution in distilled
water for only one minute.
Too much treatment will have a negative impact on fertilisation.
Then, coverslips are transferred into another falcon tube to be washed in
distilled water for one minute, twice.
Once the coverslips are washed, they are left to air dry.
Then, once the coverslip is dried, it needs to be cut in smaller sections.
So we put the coverslip in between two glass slides.
Using a diamond glass pen cutter, we cut sections of 3mm from the coverslips.
And finally, they are stored until they are used.
After cutting, three coverslip pieces will be fixed to one glass capillary.
To do so, three coverslip pieces will be placed on a supporting slide.
Then a really tiny piece of wax is delicately deposited at 5mm
from one edge of the three coverslips.
Those 5mm will be important for easily handling coverslips with forceps.
Then a capillary of 3-4cm is placed next to the wax.
Finally, the wax is melted by warming the supporting slide.
Be sure to push the capillary in the wax and then let the setup cool.
After cooling, the whole setup is placed in a plastic Petri dish by taking the
capillary with forceps.
Again, we put a slightly larger piece of wax on the capillary at the opposite
end of the coverslip.
And once again, the wax is melted by warming the supporting slide.
Be careful to take your time, otherwise the plastic Petri dish will melt before the wax.
Rapidly again, push the capillary in the melted wax, then let cool down the setup.
Finally, the mark is made outside the plate using a pen at 5mm from the end of each
coverslip piece to indicate the zone where the eggs will be aligned. It's also a visual
mark to find your way under the microscope.
The glassware setup for an experimental condition done in triplicate is completed.
To facilitate the microinjection, we need to align the eggs on the coverslip.
To do so, we produce a stretched glass pipette.
Over a moderate flame from a Bunsen burner, we warm the glass pipette
and pull it apart when the glass is getting softer.
The edge of the pipette then needs to be cut to an opening diameter corresponding
to the eggs' diameter. This can be done after under a magnifier with eggs at side.
Be sure to conserve the pipette with great care. They are reusable if we take care of them.
After all of this preparation, we can begin the experiment. We start with the alignment
of our eggs. First, our glassware setup is flooded with seawater.
Then we place the stretched pipette at the end of a personal plastic tube having
filtered tips at the edges.
Using our mouth suction under the magnifier, we delicately vacuum eggs.
Be careful to not break your pipette during those movements.
Then we carefully drop eggs on the glassware setup between the end of
the coverslip and the penmark.
Ideally, separated and aligned eggs will facilitate the microinjection a lot.
The aim of the experiment is to inject eggs with a solution containing different
concentrations of the reporter RNA of the Luciferase.
We also inject a fluorescent molecule like AlexaFluor to visualise the injection under
a fluorescent microscope.
It gives a yellow colour to the solution.
Mixed solutions are then introduced in the micropipette.
It's time to microinject.
The micropipette is mounted on the microscope,
and we place our aligned sea urchin eggs under the light.
First, the aim is to bring the tip of the micropipette in front of the eggs
so we use a motorised arm.
We used the Femtojet injection system and we can visualise on the screen or in oculars
the pipette approaching the eggs.
First we have to find the right position of the tip on the egg.
Too much on top, the egg is too soft to be injected.
Too much underneath, and the eggs break.
Then we change to the fluorescent view to see the actual injection
and then we go from one egg to another. Easier said than done, believe me.
We use the freeflow method. This procedure results in the injection of
approximately 5% of the volume of each egg. The fluorescence will be used to
relatively quantify the injection of each egg.
After injection, eggs are fertilised the same way as in the previous lab videos.
At the desired time, here one hour post-fertilisation, measurements of
luminescence can be done.
To do so, coverslips are delicately retrieved with forceps
and rapidly eggs are immerged in dolphin tubes filled with 50μL of Luciferase reagent
of Promega named One-Glo.
Tubes are then centrifuged one minute at 13,000g and the reagent containing now
the eggs is transferred in a 96-well microplate.
This plate is then inserted in a Tristar luminometer and luminescence from
each well is measured.
Here, our conditions were a dose response to the reporter mRNA concentration injected
in eggs.
The luminescence signal obtained from few microinjected eggs is standardised with
respect to the fluorescent signal. Results are expressed in relative light units (RLUs).
These values can be interpreted as the resulting Luciferase translation of our
different experimental conditions.
Using this experimental setup, we were able to detect the translation from the
injected mRNAs following fertilisation. As soon as one hour following fertilisation,
the Luciferase activity is detectable. Here, we can see the luminescence signal
corresponding to the reporter expression that is proportional to the amount of the
mRNA that was microinjected. This experiment is feasible because sea
urchin eggs are transparent and large enough to be microinjected, and posess
an efficient translational activity stimulated by fertilisation. Next steps will be now to
develop experiments that will analyse UTRs of specific mRNAs such as those encoding
for key actors like cyclin B, CDK1, etc. It will also be interesting to analyse the
effect of specific signalling pathways on specific UTR cis-regulating regions.