The overall goal of this procedure is to deliver retroviral vectors into the mouse forebrain prior to the onset of neurogenesis in that region. This method will permit transduction of germinal zone cells with genes of interest. Other viral vectors or cells could also be injected using this method.
This is accomplished by first performing a laparotomy on an anesthetized pregnant mouse, which permits access to the embryos. The second step of the procedure is to place the pregnant mouse on the injection stage under the ultrasound transducer. Then position the embryos on the plate for injection.
The third step of the procedure is to use the real-time ultrasound image to direct the microinjection needle to the injection target site. Embryos are injected in series until the desired number is completed. The final step of the procedure is to remove the mouse from the injection apparatus and close up the incisions in the body wall, muscle, and skin.
The animal is then allowed to recover. Ultimately, results can be obtained that show the location and phenotypic characteristics of infected cells through detection of a given reporter gene in tissue sections and labeling with cell type specific markers. Hi, I am Taryn Pier Fois from the laboratory of Nick Guyano in the Neurogeneration program at the Johns Hopkins University School of Medicine.
Today we will show you a procedure using ultrasound image to guide the microinjection of retroviral vectors into the mouse brain in utero at embryonic day 9.5. We use this procedure in our laboratory to study the effect of manipulating gene expression during four brain development on neuro stem and progenitor cell behavior and sulfate specification. So let's get started.
If the injections are to contain engineered viral vectors, the procedure should be performed in a biosafety two cabinet. Place an anesthetized pregnant mouse on its back. Use fine surgical scissors to make a one inch longitudinal incision through the skin at the midline.
Cut away the connective tissue located between the skin and muscle surrounding the incision. Next, make a slightly shorter incision through the abdominal muscle with forceps. Gently draw out the uterine horns.
Record the number of embryos on each side. Transfer the animal to the plexiglass injection stage. To protect the embryos.
Place most of the uterine horns back into the animal, leaving just two adjacent embryos exposed. Use a 10 centimeter Petri dish with two small thumbtack holes and a two centimeter center hole sealed with a piece of scholastic membrane to keep the embryos moist. With a pair of forceps pushed slightly through the scholastic membrane.
Lower the dish over the mother. As the dish is lowered. Grasp the uterine tissue between the two selected embryos and gently pull them through the membrane when the dish is seeded.
Secure it with two thumbtacks through the smaller holes and into the wax. Fill the dish with 37 degrees Celsius, one XPBS to keep the embryos moist. Orient the embryos in parallel to the length of the mother.
Place the injection stage under the ultrasound probe. Lower the probe to approximately two to three millimeters over the embryos. For clear ultrasound images, it is crucial that the probe head does not hit the embryos as it oscillates back and forth.
Collecting images, connect the needle to a 25 microliter micro syringe mounted in a manual infusion withdrawal pump. Place a beveled and sharpened micro injection needle into a pipette holder. Apply light pressure to a 10 cc syringe filled with mineral oil in the upper part of the stop cock to flush oil into the tubing, micro injection needle holder and needle, and draw oil into the micro syringe.
Make sure not to introduce air bubbles. Draw back an air bubble from one to two millimeters long into the needle. To maintain separation between the mineral oil and the virus, use the withdrawal pump to carefully load the needle with virus solution.
Draw the solution smoothly and slowly and pay attention to avoid clogs. Place the loaded needle into the microm manipulator position in the injection area. Taking care not to break it on the embryos or the ultrasound probe.
Move the injection needle into position one to two millimeters away from the uterine wall. At about a 45 degree angle, images generated by the ultrasound probe are visualized on a video monitor. The needle tip appears as the brightest spot on the screen.
Make sure that the oscillation direction of the probe is parallel to the length of the needle. Using the ultrasound image on the video monitor position the embryo such that the target region, in this case the four brainin ventricle, is readily visible and accessible to the needle. Line up the target brain region with the direction of needle advancement.
Use the micro manipulator to bring the tip of the needle to just touch the uterine wall. Quickly jab the needle forward to penetrate the uterine tissue. Multiple similar jabs may be necessary to penetrate the amniotic sac and the brain inject the embryo with the appropriate volume of virus.
In a successful injection, a particulate haze enters the ventricle and the ventricular space may expand slightly, withdraw the needle back approximately five millimeters before injecting the next embryo. When both embryos have been injected, use forceps to pull the next two embryos through the scholastic membrane and return the injected embryos into the abdomen. Once the desired number of embryos have been injected and pushed back into the abdominal cavity, move the mouse out of the injection stage and onto an absorbent pad.
To close the incision, allow the animal to recover in a clean warmed cage. This screen capture of a real-time ultrasound image from an E 9.5 mouse embryo shows various anatomical features that can be used to guide injections. Here V is the ventricle as the amniotic sac and uw the uterine wall.
The injection needle is the bright spot on the right of the image. In this image, an embryo injected with A-P-L-A-P expressing virus shows PLAP positive virally infected cells within the developing brain. Several weeks post-infection reporter expression allows examination of cell morphology and cell position, as well as a clonal analysis.
We've just shown you how to perform ultrasound guided micro injection of retroviral vector into mouse embryos in utero at embryonic day 9.5. When doing this procedure, it's important to remember to move quickly as survival is more likely the less time the mouse is being manipulated during the surgery. It's also important to remember to be careful when using biohazardous materials such as retroviral vectors.
So that's it. Thanks for watching and good luck with your experiments.
