The overall goal of this procedure is to perform high throughput micro injections of sea urchins zygotes. To do this spawning is induced in the sea urchins and male and female gametes are collected. The eggs are then deed using acidic seawater and immobilized in a row on Petri dishes.
Sperm is then added to the eggs to fertilize them and micro injection of the desired Mr.N.A loss of function, reagents, or proteins is performed following microinjection. The three A T water containing sperm is exchanged for fresh seawater. These embryos can be examined for their morphology and the temporal and spatial expression of specific RNA and proteins.
The main advantage of this technique over existing methods like electroporation, lip perfection, microparticle, bombardment, and transduction, is that it enables delivery of any solution with 100%efficiency using minimal reagents. This method enables efficient and reliable microinjection of 100 to 400 x on a single dish of thousands of X per experiment, providing a high throughput method for downstream analysis. Prior to performing this experiment, prepare all stock solutions and equipment needed.
See the accompanying document for recipes, a mouth pipette will be needed for gentle handling of the eggs. To prepare one begin by holding a glass micro pipette with both hands over an open flame as the glass begins to melt, carefully pull the ends of the pipette in opposite directions until the pipette separates into two halves. Next, connect one pulled pipette to plastic tubing 50 to 70 centimeters in length.
Apply paraform to create a tight seal. Insert a sterile P 20 or P 200 filtered tip into the other end of the tube for use as the mouthpiece finally clip the end of the glass micro pipette with scissors to adjust the diameter of the tip, the optimal diameter will slightly exceed the diameter of the egg, which is roughly 80 micrometers. Next, prepare protamine sulfate coated dishes.
Lay out 2060 millimeter by 15 millimeter polystyrene petri dishes on the bench top and remove dish lids. These will also be used as dishes is pour 1%protamine sulfate solution hereafter, referred to as PS solution into each dish. Add just enough to cover the surface and incubate for at least two minutes following the incubation.
Remove the PS solution and save it for later use. The leftover PS solution can be reused many times within three months when stored at four degrees Celsius. Place the PS treated dishes in a beaker filled with distilled water.
Then place the beaker under running distilled water for at least 10 minutes. The PS coated dishes can be used immediately or air dried for storage. Cover them to prevent dust accumulation.
They can be stored at room temperature for up to one month. To fabricate injection capillaries, use a needle puller. The settings of the needle puller need to be empirically determined as stated in the manuscript.
To spawn sea urchin, remove an animal from the tank using a net and place it on a paper towel. Then pick up the animal and holding it tightly. Shake it.
Alternatively, perform an emmic injection as follows. After removing an animal from the tank, hold the animal in one hand and use a 20 gauge needle connected to a 10 milliliter syringe to inject one milliliter of 0.5 molar potassium chloride into the perial membrane, the only soft part of the animal. After one to 10 minutes, the animal sex will become apparent.
Males will begin to release white sperm and females will begin to release yellow eggs. If the animal is male, collect the sperm dry from the surface of the animal using a pipette to transfer it into a 1.5 milliliter tube. Dry sperm can be stored at four degrees Celsius for a week without significant loss of biological function.
If the animal is a female, immerse the gno pores in a beaker full of seawater to collect its ga meats. The eggs released from gno pores will settle down by gravity. After spawning, filter the eggs from debris such as animal spines by pouring the seawater containing the eggs.
Through an 80 micron nylon filter mesh into a clean beaker on ice. To deje the eggs transfer up to one milliliter of eggs to 100 milliliters of acidic seawater in a plastic beaker, gently swirly eggs and allow them to settle on ice for 10 minutes. Following treatment carefully pour off acidic seawater.
Then add fresh sea water and let the eggs settle down on ice.Again. De jelled eggs can be stored on ice for up to six hours without affecting fertilization. Immediately before performing micro injections, pour three to four milliliters of one millimolar, three a TC water into each PS coated dish.
The X should be all kind before the injection hospitalization Problem might occur if there is a prolonged exposure to three 80 c water more or onic adhesives, such as protein sulfate can be toxic to the X.So exposure time should be linked, especially if the fertilization animal Have been removed. Secure the tip of the mouth pipette with the teeth and aspirate a small amount of sea water. This will increase control over the eggs when they're aspirated.
Next, position the micro pipette tip near the eggs and gently aspirate several hundred eggs, confining them within the glass micro pipette. Next, while moving the tip in a straight line, gently blow them out of the pipette into the dish to form a row. Using a glass pipette, make a scratch on the dish near the line of road eggs.
This scratch may be used later to break the injection needle. To adjust the flow of solution as needed during the micro injection process. Turn on the micro injection station here.
A femto jet injection system is used carefully using a pair of forceps. Mount the needle capillary on a needle loading holder. Avoid touching the end of the needle to prevent contamination of the needle opening.
Using a micro loader tip, load it with 0.5 to 1.0 microliters of sample injection solution. Place the dish with road eggs on the microscope stage and position the dish so that the eggs are aligned vertically. When viewed through the eye pieces, bring the eggs into focus.
Carefully mount the loaded needle onto the needle holder of the micro injector and adjust the position of the needle so that the tip is perfectly in Focus in the middle of the field of view. Next to clean the needle, depress the button to apply the maximum pressure viewing through the microscope. The solution will be seen as it is expelled from the needle.
Adjust the pressure to be in the range of 90 to 360 HP for injection with the compensation pressure at approximately one third of the injection pressure. Next, inject an unfertilized egg to examine the flow of the injection set up. Using a joystick micro manipulator, direct the tip of the injection needle to the unfertilized egg.
To facilitate needle entry, create a slight trembling by gentle tapping of the stage with a hard object such as a screwdriver. Press on the foot pedal to inject the egg. If the injection bolus is not visible, bring the needle to the scratch mark.
Gently tap the needle tip to the scratch mark to break the tip of the injection needle. The slightly wider tip opening will enable easier solution flow and the sharp edge of the broken tip facilitates needle entry. Fertilize the eggs by adding sperm immediately after the fertilization envelope becomes visible.
Start injections using the micro manipulator. With your right hand, press the tip of the needle against the fertilized egg, causing a slight indentation almost simultaneously. Gently tap the microscope stage with the screwdriver held by the left hand.
Move along the line of road zygotes using the stage controller and inject as many zygotes as possible. The injection bolus should not exceed one fifth of the zygote. After 10 to 15 minutes, the newly fertilized eggs will become hardened and can no longer be injected.
Remove the dish from the stage and using a plastic pasta pipette carefully aspirate the three A TC water containing sperm. Do not let the zygotes dry and quickly add fresh pre chilled sea water. Cover the dish incubate at 15 degrees Celsius.
Following injection. Monitor the embryos for phenotypic or molecular changes as appropriate for the experiment to determine whether injection of reporter constructs affected development. GFP and m cherry reporter constructs were in vitro transcribed and micro injected into the newly fertilized eggs.
The injected embryos shown here can be clearly distinguished from those not injected, which are indicated by the arrows. Embryos were incubated at 15 degrees Celsius for 24 hours to the blastula stage and imaged as can be seen here. The injected embryos which are colored have the same morphology as non injected embryos, which are gray.
This suggests that injection of reporter constructs does not lead to any developmental defects. After watching this video, you should have a good understanding of how to micro inject the urchin. Zygotes Microinjection is a valuable tool for research in genetics, cellular, and developmental biology.
Using this technique, the sea urchin community has made a significant contribution to our understanding on of developmental pathways such as sulfate specification, gene regulation, gene expression, and biochemical pathways that are underlying the developmental biology.
