The overall goal of this modified procedure is to increase chimeric production without incurring additional costs or long training periods. This method can help laboratories become more efficient in chimera production, especially in cases where the ES cells are less than optimal. The main advantage of this technique is that it requires no additional equipment and no specialized training.
Demonstrating the procedure today will be Thomas Hagler. He's a biologist and graduate student who works in the core lab. To harvest the embryos, blot the uterus on a lab tissue to remove the excess blood and place the organ in a new 60 millimeter dish under a low magnification dissecting microscope.
Using dissecting forceps to carefully and gently hold the uterus near the oviduct, insert a 25 gauge needle attached to a 10 milliliter syringe containing 10 milliliters of blast collection medium into the center of the uterus just beyond the tips of the forceps in as parallel a plane as possible. Apply pressure to the forceps to secure the needle within the uterus and gently flush approximately one milliliter of medium through the uterine horn. When all of the blastocysts have been expelled, assemble the mouth pipette apparatus and use the mouth pipette to transfer the individual embryos into single drops of blast collection medium in a second 60 millimeter dish.
Then wash each embryo in a second drop of blast collection medium and transfer the embryos into a four-well dish until their injection. To begin, use a microinjector to apply vacuum to the injection needle. Place a dish of ES cells under the injection microscope where the injection needle and embryo holder pipette are attached.
It is essential to the success of the injection to take the time to properly setup the microscope. This includes the proper amount of media in the needle and the correct alignment of the z-axis. Use a microinjector to apply vacuum to the injection needle.
Keeping as little space between the cells as possible, draw in smoothly surfaced small to medium sized embryonic cells without obvious defects from the dish of embryos. After collecting at least 15 cells, position an embryo near the opening of the holder. Apply vacuum to secure the embryo to the holder and move the holding pipette up or down to adjust the z-axis until the embryo is just touching the slide.
Fine focus on the zone of pellucida and adjust the z-axis of the injection needle until the embryonic cells near the tip of the needle are in focus. If needed, use the injection needle to gently roll the blastocyst until the inner cell mass is in the appropriate position for injection. Using a firm but controlled push, insert the injection needle through the zone of pellucida into the inner cell mass and apply a slight pressure to the microinjector of the injection needle to help keep the blastocoele from collapsing.
Once the needle has penetrated the blastocoele, apply pressure to the microinjector to transfer all of the embryonic stem cells into embryo, pausing briefly as the bevel of the needle starts to exit the embryo to allow the pressure in the embryo to return to normal while still retaining the cells. When all of the blastocysts have been injected, use a nonsurgical embryo transfer device to deliver the embryos to embryonic stage 2.5 Swiss Webster mice according to the manufacturer's instructions and house the mice individually after each transfer through delivery and subsequent weaning. In this representative experiment, the pregnancy rates were unaltered between the trans inner cell mass injected and control traditionally injected groups and an equal number of pups survived to weaning from each group.
Notably, while the number of mice at weaning remained unchanged, a significantly higher number of pups from the trans inner cell mass injected group displayed coat-color chimerism compared to the control injected group. The best part of this technique is that it only requires a simple change in the orientation of the embryo to facilitate greater production in chimeras. Proper setup of the microscope is essential.
If the microscope and manipulators aren't setup properly and maintained well, the injection process will not work. If you're new to embryo injections, repetition is the only way to learn. For beginners, embryo handling tends to be the most difficult part of the procedure.
Being able to move embryos efficiently from one dish to another without losing any is the most crucial part of the experiment. After watching this video, you should have a good understanding of how a simple change to your standard injection technique can help you become more efficient in chimera production.
