Male and female zebra fish are mated in a breeding cage in which fertilized eggs fall to the bottom where they can be easily collected. The eggs are then lined up against a microscope slide in a Petri dish. Next, the loaded injection needle is clipped and injection settings are manipulated, such that the injection bolus is a desired size when measured with a micrometer.
Finally, eggs are injected one at a time down the line. Hi, I'm Jonathan Rosen from John MA's Laboratory in the Department of Cardiology at Children's Hospital Boston. And I'm Michael Sweeney, also in the VY lab.
Today we're gonna demonstrate how mRNA or morph Fello can be injected into zebrafish embryos. We use This procedure in our laboratory to study the effects of knockdown or overexpression of particular gene products. So let's get started.
To begin, we'll need to set up the fish and breeding tanks with dividers in place the night prior to injection to increase total egg production. Fish can be set up in a ratio of two females to one male if desired. The next morning after the room lights are turned on, pull the dividers from several tanks and allow for approximately 20 minutes of undisturbed mating time Using a strainer, collect the eggs from the breeding cages and rinse them with egg water before pouring them into a Petri dish.
With egg water, fish can be regrouped in larger tanks to produce additional rounds of eggs for injection. Adjust the timing of egg collection to allow for the maximum numbers of eggs to be produced without letting them pass the single cell stage. Finally, place a microscope slide in the inverted lid of a 100 millimeter Petri dish.
Use a transfer pipette to line up the eggs against the side of the slide forming a single column, and then remove excess egg water from the slide by pressing a Kim wipe against the side opposite the eggs. Next, we'll prepare the micro injection needles using a micro pipette polar. We'll pull a one millimeter outer diameter glass capillary into two needles and store these needles in a 150 millimeter Petri dish By laying them over silly putty ramps, needles can be pulled in advance.
We use the following settings. Heat 6 45 pole 60 velocity 80. Load the wide end of the needle with three microliters of injection material using a micro loader pipette.
Shake the bolus toward the needle tip until there are a few or no bubbles remaining. Next, we'll turn on the air source and micro injector. Insert the needle into the micro injector and ensure a tight seal within the housing.
Check that the micro manipulator is in a proper position to allow for a wide range of movement and adjustment. Bring the needle tip into the plane of view of the microscope. High off the stage and focus on the thinnest region of the tip.
Use a pair of sharp forceps to pinch off the needle at a point which leaves the needle narrow enough to pierce the corry on and yolk, but still capable of delivering a consistent bead size. To calculate the volume of each injection, we'll use a drop of mineral oil on a micrometer, insert the needle into the oil and depress the foot pedal to inject a bead of solution into the oil. Monitor the size of the bead while trimming the needle and adjusting the injection pressure is needed when injected into the oil.
A bead with a diameter of 0.1 millimeter contains 500 picoliters of injection material. Injection volumes of 500 picoliters or one nanoliter are typically used. Ideal injection volumes will fill approximately 10%of the egg volume.
The quality of the needle tip is crucial to both the ease of injection and the quality and consistency of the results. Now it's time to inject to begin the injection procedure. Ensure that the embryos have not developed past the four cell stage.
Ideally, embryo should be at the one cell stage. Next, lower the needle toward the column of eggs, pierce the surface of the corion and enter the yolk in one smooth stroke while watching. For any crushing or tearing of the yolk sack, inject the injection material into the yolk.
Avoid injecting air bubbles and watch out for embryos that are visibly damaged by the injection working down the line. Adjust the pressure is needed to maintain a consistent bead size and using a pipette tip, remove eggs which look unfertilized or are destroyed during the injection process. After completing a column of eggs, use a gentle stream of egg water to move the injected eggs into a clean Petri dish and repeat as necessary.
Keep several unin injected embryos as a control. Later on during the first day, remove dead embryos and record the number of injected embryos. Replace the egg water in the dish periodically to reduce the chance of infection.
Note that depending on what is being injected, embryos may survive at a lower rate than their unin injected siblings. Fortunately, it is easy to inject huge numbers of them, so this is rarely a problem. Now we'll show some representative results of both morpho injection and mRNA.
The unin injected control at 48 hours post fertilization looks normal as expected. However, embryos injected with the morpho HG E three i three eeg FFR one, which knocks down HG isoforms containing the first of two EEG flike repeats exhibit, brain edema, injection of heart glass. Mr.NA also produced an obvious phenotype at 24 hours post fertilization.
The heads of the unin injected controls looked normal. Conversely, some of the embryos injected with the M NNA exhibits opia, We've just shown you how the injection of morphos or mRNA during the one cell stage can increase or decrease the expression of specific genes during later development. When doing this procedure, it's important to remember that too.
High concentration of either mRNA or morpho can cause non-specific lethality in the embryos with the morpho. Each one has to be tested empirically, but typically concentrations of between 200 and 500 micromolar knockdown gene activity specifically without causing overall lethality. So that's it.
Thanks for watching and good luck with your experiments.
