This method allows rapid in vivo screening in mice, of the effect of a transgene, and is specifically adapted to perform gain or loss of function studies, as well an answer and DNA motive mapping. The main advantage of this technique, is that the prediction use of transgenic animals are above 70%of newborn and can be applied to any mouse strains including transgenic animals. After euthanizing the mice, collect the oviducts.
To begin the surgery, use scissors to make a large horizontal incision into the abdominal cavity. Then, locate the oviduct between the uterus and the ovary. Next, use curved forceps to remove the mesometrium and the membrane with the prominent blood vessels.
Then, separate the oviduct from the ovary and dissect the oviduct from the ovary. Then, tug on the oviduct and cut away from the uterus. Transfer all the collected oviducts to M2 medium at room temperature.
When handling the oviducts, be careful to avoid touching the swollen ampulla loaded with fertilized eggs. Next, add a 100 microliter drop of hyaluronidase working solution to a 10 centimeter dish. Then transfer the two oviducts into it, to remove the cumulus cells from the eggs.
Under a stereomicroscope, use a pair of insulin syringes to secure the oviduct while tearing up the ampulla and dispersing the fertilized eggs. Then, use a prepared glass pipette with tubing and a mouthpiece to aspirate all the eggs. Using this tool, transfer them between six drops of M2 medium to wash off the surrounding cumulus cells.
Then, transfer the washed eggs into a 10 millimeter well, in a four well plate, filled with preheated with M16. Then, transfer the dish to a humidified 37 degree Celsius incubator. In preparation, centrifuge the prepared lentiviral vector suspension at 160 g for two minutes, to pellet the debris often present in lentiviral stock.
In a class two safety cabinet, transfer the supernatants to a new 0.5 milliliter tube. Next, load one microliter of supernatant, into an injection pipette. Then, attach the injection pipette to the right side micromanipulater.
And attach the holding pipette to the left side micromanipulator. Now, dispense eight microliters of M2 medium in the center of the depression slide. And cover with light parafin oil to avoid evaporation.
Then, place 20 eggs into the shallow locations in the drop without creating bubbles. Next, make sure that the tip of the injection pipette is open. If not, tap the injection pipette with the holding pipette to open it.
Then, set the microinjector for 20 second injections. Now, using the holding pipette and manual pressure aspirate a fertilized egg that contains two pronuclei and two polar bodies. Then, using the injection pipette inject the perivitelline space of the egg.
Do not touch the plasma membrane. Adjust the pressure so that the perivitelline space is filled with solution. The pressure should not exceed 600 hecto pascals.
Immediately after injecting the 20 eggs, transfer the batch into a bath of preheated M16 medium. And incubate them for at least 30 minutes before implanting them. 16 hours after mating normal females to males with vasectomies, check for copulation plugs.
Use these females for the implantation. Next, make implantation pipettes with internal diameter of about 150 microns. And with flame polished tips that are about four to five millimeters long.
Load a pipette with embryo tested, light parafin oil to just above the shoulder point. Then, aspirate a small air bubble and further load the pipette with M2 medium. Next, aspirate a second air bubble.
Now, draw up the embryos into the pipette one behind the other, minimizing the volume of medium between the embryos. Finish, by aspirating a very small drop of parafin oil just one embryo wide. Preparation of the reimplantation pipette is key and needs a lot of practice.
Eggs in the pipette must be aligned with practically no medium in between them. Next, after anesthetizing the mouse and confirming the anesthesia with a toe pinch Shave off two, two centimeter strips of hair along the spinal cord at the level of the last rib. Now, place the mouse on a heating pad on a sterile field and drape it, leaving a window to the exposed skin.
Next, sanitize the exposed skin with alternating scrubs of iodoprovidone and 70 percent ethanol. For the implantation, first make a one centimeter transverse incision, and then slide the skin laterally until the orange colored ovary is visible through the body wall. Next, make a five millimeter incision with the fine scissors and pick up the fat pad with an atraumatic bulldog clamp.
Once the pad is gripped, pull out the ovary, the oviduct and the top of the uterus. Next, locate the ampulla and make a hemisection with vannas scissor, on the oviduct segment that links the ovary to the ampulla. Then, introduce the transfer embryo pipette and eject the eggs.
Stopping at the first air bubble after the eggs have been released. Have the required materials prepared to repeat the procedure on the second oviduct. And once completed, close the skin up with wound clips.
Immediately after closing the skin, intraperitonealy inject analgesic and place the animal in the recovery chamber with a 39 degree Celsius heating element. Monitor until it is fully awake. Transgenic animal were generated using the presented method.
A high titer lentiviral vectors were produced using a 2.3 kilo base enhancer for Neurogenin-3 to drive eGFP. Or as a control, a fragment of chromosome six to drive eGFP. Integration into embryos was fairly successful.
From the embryos, the pancreatic bud was sectioned and immunostained to look for localized expression of eGFP. Over 90 percent of the vectors, with the Neurogenin-3 enhancer, expressed eGFP in Neurog3 expressing cells. However, none of the integrated control vectors expressed eGFP in the pancreatic bud.
Transgene integration sites were evaluated by quantitative PCR, using the CDX2 gene as a control. The average number of integration sites was about 20 using the Neurogenin-3 construct, or 10 using the control construct. Curiously, the viral transduction titer used for the Neurogenin-3 construct, was about double of the control vector.
Suggesting a relationship between the transduction titer and the number of integrations. After watching this video, you should have a good understanding of how to generate large numbers of founder transgenic embryos using perivitelline injection of lentiviral vectors with high efficacy. Once mastered, this technique can produce up to 50 founder animals within a single day of microinjection, if it's performed properly.
Following this procedure, other methods like titer pronuclear DNA injection can be performed, in order to establish transgenic mouse lines. Don't forget that working with lentiviral vectors is subjected to authorization from your local GMO committee. These vectors can be extremely hazardous and precaution should always be taken while performing this procedure.
