Transgenic animals are fundamental to modern biomedical research as they allow studies of gene function in living organisms. Various methods of genetic modifications have been applied in animal studies. Here, we present a widely accessible and effective technique that uses lentiviral vectors as a tool for transgene incorporation into the genome of a rat embryo.
After human chorionic gonadotropin administration, mate five-week-old Wistar female rats one-to-one with sexually fertile three to 10-month-old Wistar males. At eight to 10 a.m. the next morning, check the females for the presence of a vaginal plug and harvest the oviducts from the females with a mating plug by no later than 10 a.m.
into a collection dish containing pre-warmed M2 medium. When all of the oviducts have been collected, transfer the tissues into a 35 millimeter dish containing pre-warmed M2 medium supplemented with 0.5 milligrams per milliliter of hyaluronidase from bovine testes and place the dish under a stereo microscope. Use fine forceps to open the walls of the oviduct and press the ampulla until the embryos are freed.
To facilitate the release of the embryos from the cumulus cells, use a glass transfer pipette connected to a mouth-operated aspirator tube to gently pipette the embryos up and down. When all of the embryos have been released, wash the cells a few times in fresh M2 medium to remove the hyaluronidase and cellular debris. Transfer the embryos into individual 50 microliter drops of pre-equilibrated M16 medium covered by mineral oil in a 60 millimeter dish.
Then place the dish into a humidified 37 degree Celsius incubator with a 5%carbon dioxide atmosphere until their injection. For lentiviral vector microinjection under the zona pellucida of the one-cell stage embryos, use a pipette puller to prepare microinjection borosilicate glass capillaries with a filament. After every time the filament is changed or a new glass capillary is used, run a Ramp test to set the optimal parameters.
Next, use a microloader tip to load approximately two microliters of freshly thawed lentiviral solution per microinjection pipette under a biosafety laminar flow hood. Add a 100 microliter drop of M2 medium to the center of the lid from a 60 millimeter Petri dish. Cover the medium with mineral oil and mount the holding pipette and the virus loaded microinjection capillary onto a micromanipulator.
Place the microinjection dish under an inverted microscope and transfer 15 to 20 one-cell stage embryos into the M2 drop in the microinjection dish. Capture one embryo with the holding pipette and use the 400 times magnification and the glass capillary to inject the lentiviral solution under the zona pellucida into the perivitelline space holding the capillary under the zona pellucida for a moment after the virus has been delivered. When all of the embryos have been injected, use a fine pipette to return the injected cells to the culture dish and return the dish to the cell culture incubator until their implantation.
To prepare the foster mothers for the embryo transfer, mate sexually mature Sprague-Dawley females with vasectomized males and check the females the next morning for a vaginal plug. After confirming a lack of response to pedal reflex in the anesthetized females with a viable plug, apply ointment to the animal's eyes and shave the fur from the back of each animal. Place the first rat in the prone position on a clean surface on a heating pad under a surgical microscope.
Cover the rat with a sterile drape with a small hole cut over the lower back and make an approximately two centimeter skin incision parallel to the lumbar vertebral column. Use sharp scissors to make a cut in the abdominal wall and use forceps to grasp an ovarian fat pad. Pull the ovary and oviduct out of the abdominal cavity onto a piece of 0.9%sodium chloride soaked gauze.
Load M2 medium, three bubbles of air, and the embryos into a transfer capillary. Use microscissors to make a small incision into the oviduct between the infundibulum and ampulla and insert the tip of the transfer pipette into the incision. Gently expel the embryos and air bubbles into the oviduct, then use blunt forceps to place the reproductive tract back into the abdominal cavity.
Suture the abdominal wall with polyglycolic acid absorbable sutures and close the skin incision with wound clips and place the animal in a clean cage on a warming plate with monitoring until full recumbency. To vasectomize potential mates, after preparing the male rat for surgery as demonstrated, use 70%ethanol and a surgical scrub to disinfect the skin over the testes. Gently press the abdomen to expose the testes in the scrotal sac and cover the rat with a sterile drape with a small hole over the surgical site and use scrotal scissors to make a 0.5 centimeter incision in the middle of the sac.
Carefully push the testis to the left and locate the vas deferens between the testis and midline as a white duct with a single blood vessel. Use watchmaker's forceps to gently pull the vas deferens out of the scrotal sac and holding the vessel with forceps, use fine scissors to remove a one centimeter fragment from the duct. Repeat the procedure for the second testis as just demonstrated and close the skin with polyglycolic acid absorbable sutures.
Then place the rat into a clean cage on a warming plate with monitoring until full recumbency. In this representative experiment in which single embryos were injected multiple times, eight rats were born from embryos that were injected two times, three of which were confirmed to carry the transgene. One of the founders did not transfer the transgene to offspring and three foster females were used for each experimental setup.
The chosen approach allowed the generation of stable transgenic rat lines that expressed the TDP-43-eGFP fusion protein under the control of the neuronal synapse in one promoter throughout the central nervous system. Lentivirus-based transgenesis resulted in a single copy insertion of the transgene as demonstrated by quantitative PCR. When this method was used for other lentiviral vectors, an approximately 90%survival rate was observed.
The percentage of embryos that survived the pronuclear injections was significantly lower. Overall, these results suggest that pregnant female rats can be used as foster mothers with a comparable efficiency. Notably, the numerical data that were analyzed for individual rounds of microinjection indicated that the effectiveness of implantation depended directly on the number of injections into one embryo and indirectly on the viral load.
The use of lentiviral vectors guarantees the genomic integration and transmission of the transgene and facilitates a high survival rate of the micromanipulated embryos even when multiple subdermal injections are performed. This procedure may be effectively applied to other species and can be considered an alternative for conventional transgenesis.
