This protocol helps us determine how alterations in local biomechanics, tissue architecture, paracrine environments and juxtacrine interactions influence cellular phenotype. This technique allows us to explore classic paradigms and experimental embryology without causing the large scale tissue insults that normally arise in traditional graphing experiments. To find the right pressure for injection, it's helpful to start at a lower injection pressure and increase incrementally until a steady flow of cells is achieved.
Visualizing this technique helps in achieving a direct understanding of how the injection equipment needs to be positioned in relation to the target tissue. Demonstrating the procedure will be two members of my laboratory, Trevor Henley and Kandace Thomas. 24 hours before the implantation, pull glass capillaries on a micropipette puller according to standard protocols and dip the capillaries in siliconizing agent to coat the external surfaces of the needles.
Next, load 5 to 10 microliters of siliconizing agent into a microinjection pipette tip and place the tip in the wide end of one externally coated pulled glass capillary. Position the pipette tip as close to the glass needle tip as possible and eject the siliconizing agent while slowly retracting the loading pipette to minimize air bubbles within the needle. Leave the siliconizing agent in the glass needle for 10 minutes before using a new loading pipette to aspirate the solution.
Then dry the needles in a fume hood overnight. For host embryo preparation, incubate fertile chicken eggs in a horizontal orientation in a humidified incubator at 38 degrees Celsius and use angled forceps to make a greater than one millimeter diameter puncture in the bottom of the flat end of each egg along the egg equator. Insert an 18 gauge needle attached to a 10 milliliter syringe into the puncture to remove about five milliliters of albumen.
And apply transparent tape to the top of the egg shell. Score the taped region along the top of the egg with angled forceps and use curved tenotomy scissors to cut an approximately 2.5 centimeter window in the taped section of the egg. Inspect and stage the embryo based on the criteria established by Hamburger and Hamilton and use a one millimeter syringe equipped with a 32 gauge needle to inject about 200 microliters of a one to five dilution of India ink in HBSS beneath the embryo.
Then seal the puncture with transparent tape and add one milliliter of HBSS drop-wise onto the embryonic disc before sealing the windowed shell with paraffin film for placement of the egg back into the humidified incubator. When the embryos reach Hamburger and Hamilton stage 19, rinse the silicone-coated glass capillaries with deionized water and allow the capillaries to dry for three to four hours at room temperature. In the mean time, transfer an embryo from one egg into a 100 by 15 millimeter Petri dish containing sterile room temperature HBSS and place the dish under a stereo microscope.
Using forceps, tenotomy scissors and a micro spatula, isolate the entire embryonic heart from the embryo and isolate the atria from the heart. Place the atrial tissue in a sterile 1.5 milliliter microcentrifuge tube containing one milliliter of HBSS on ice. When all of the donor tissue has been collected, pellet the tissues by centrifugation in a fixed angled microcentrifuge.
For donor tissue digestion, re-suspend the pellets in one milliliter of pre-warmed 05%trypsin EDTA for a 15 minute incubation in a shaking heat block at 300 rotations per minute. At the end of the incubation, pipette the digestion solution several times to break up any remaining tissue and pellet the cardiac lysate by centrifugation. Re-suspend the pellet in one milliliter of trypsin neutralizing solution for another centrifugation followed by re-suspension in 400 microliters of red fluorescent dye solution.
After 20 minutes at 37 degrees Celsius, pellet the cells by centrifugation for one to three washes in one milliliter of HBSS per wash. Then re-suspend the labeled cells at a five times ten to the fourth cells per microliter concentration in fresh HBSS. For in vivo injection of the donor cells, backload the cell suspension into a dry, silicone-treated glass capillary pipette as demonstrated and mount the pipette into the pressure microinjector apparatus.
Transfer each host embryo to be injected from the humidified incubator into an egg holder under a fluorescent stereo dissecting microscope. And use sterile fine forceps to open the vitelline membrane. Make a 5 to one millimeter incision in the pericardium and position the microinjecter such that the tip of the microinjection needle penetrates the target tissue.
Set the microinjector to apply single pulses of 5 seconds in duration and ranging from 100 to 400 hectopascals in pressure. And pressure-inject the cells. It is critical to verify a successful cell implantation by imaging the fluorescent signal prior to resealing the egg.
The embryo can also be photographed to document this procedure. When all of the cells have been injected, retract the microinjector apparatus and remove the egg from the holder. Then add one milliliter of warm HBSS drop-wise onto the embryo.
Seal the egg with transparent packing tape and return the egg to the humidified incubator for 24 hours post-implantation. Here, the heart and surrounding tissue of a host embryonic heart after donor atrial myocyte implantation is shown. In this representative experiment, the donor cells were microinjected into the proepicardium of a host embryo of a similar stage.
Optical sectioning using a confocal microscope revealed that the only cardiac muscle marker positive cells within the proepicardium were the focally implanted fluorescent red positive cells. Take care not to over-manipulate the recipient embryo as ruptures in the heart and local vasculature caused by the injection needle can result in reduced viability. Following this procedure, a variety of downstream assays can be performed, including immunohistochemistry, in situ hybridization and cell sorting.
The siliconizing agent is extremely flammable and acutely toxic. It should always be handled with care and proper personal protective equipment inside a fume hood.
