Intraventricular Injection of Retrovirus

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The procedure for making injections of retrovirus into the telencephalic ventricles of embryonic mice and rats will be described. The basic procedures can be adapted to inject retrovirus into other structures, such as the eye (15) or olfactory epithelium (16,17). Intraventricular injections of retrovirus can be made most readily between embryonic d 11 and 15 of mice and between embryonic d 13 and 17 of rats. Injections earlier and later than the stated times are difficult because the uterine membranes are somewhat opaque, making it difficult to resolve the enclosed embryo. Furthermore, at earlier times the embryo, as well as its brain is very small and difficult to aim for with the tip of a micropipet (see Note 28). Nevertheless, the particular question under investigation will determine the precise developmental age at which the rodent embryo is injected with recombinant retroviruses.

1. Sterilize the instruments and work area: Place the instruments in a tray or beaker with 70% alcohol for approx 30 min; subsequently allow them to air-dry on sterile gauze or paper. Clean the surgical work space with 70% alcohol and allow the surface to air-dry.

2. The use of a pneumatic picopump simplifies the procedure for delivering retrovirus into the chosen location of the developing embryo, and lateral ventricles of the forebrain in particular (see Note 29). Turn on the picopump and set the flow rates for the vacuum and the nitrogen gas tank. Attach a pipet to the electrode holder connected to the picopump. The pipet tip should be broken with forceps; the optimum diameter will need to be determined by the investigator. However, the pipet tip needs to be rigid enough to penetrate the uterine membranes and fetal skull without breaking, but sufficiently thin to prevent untoward tissue damage. Pipets that gradually taper to a sharp point usually work the best. To check the rate of intake, outflow, and hold pressure of the picopump before filling the pipet with retrovirus, first draw up colored water through pipet. Adjust rates if necessary, according to manufacturers suggestions (see Note 30).

3. Anesthetize the pregnant rat or mouse by an intraperitoneal injection of chloral hydrate (or another suitable anesthetic) (see Note 31). After the pregnant dam is completely anesthetized and no longer responds to forelimb pinch or displacement of the eyelid, immobilize the dam with limbs extended on the surgery platform. The limbs should be loosely extended and taped in a fixed position on the platform or secured in some other comparable way (see Note 32).

4. Shave the abdomen of the immobilized dam from the pubic bone to the xiphoid process (see Note 33). Clean the region of the shaved skin with 70% ethyl alcohol followed by sterile PBS. In circular movements, wipe from the center of the surgical field to the margins with both the alcohol and PBS.

5. After illuminating the abdomen with the fiber optic light, gently lift the skin at the midline of the anesthetized pregnant dam using dull or small toothed forceps. Incise the superficial skin layer of the abdomen (see Note 34), using a #10 scalpel blade, along the linea alba, which will be apparent as a longitudinally running

Exposure of Uterus

Injection of Retrovirus

Micropipette

Uterine Swellings

Embryo

Exposure of Uterus

Injection of Retrovirus

Micropipette

Uterine Swellings

Embryo

Mice Atelectasis

Cerebral Ventricle

Micropipette

Fig. 1. Method for introducing recombinant retrovirus into the mid-gestation rat embryo. (A) To expose the uterine horns a midline incision is made through the skin and muscle layers of the abdomen of an anesthetized rat. In this figure three uterine swellings of the right uterine horn are diagrammed. In utero the orientation of the embryos is variable. (B) An enlargement of one uterine swelling illustrating the placement of a micropipet in the right lateral ventricle of an embryonic d 16 rat. Shaded area represents the placenta. (Reproduced with permission from Luskin, 1992.)

Cerebral Ventricle

Micropipette

Fig. 1. Method for introducing recombinant retrovirus into the mid-gestation rat embryo. (A) To expose the uterine horns a midline incision is made through the skin and muscle layers of the abdomen of an anesthetized rat. In this figure three uterine swellings of the right uterine horn are diagrammed. In utero the orientation of the embryos is variable. (B) An enlargement of one uterine swelling illustrating the placement of a micropipet in the right lateral ventricle of an embryonic d 16 rat. Shaded area represents the placenta. (Reproduced with permission from Luskin, 1992.)

white line where the external and internal oblique muscles of each side of the body meet. Extend the opening anteriorly and posteriorly; do not extend the opening rostral to the rib cage or more caudal than the position of the bladder. The smaller the abdominal opening, the better.

6. Use dull or small toothed forceps to lift the exposed abdominal muscles away from body, and then with care use scissors to make a small midline, longitudinal opening in the muscle wall. Use an index finger or blunt instrument to ensure that the abdominal organs are not adhered to the muscle wall before extending the incision rostrally and caudally about the same extent as the overlying skin layer was opened.

7. Surround the entire surgical field with a bed of gauze. Open 3 x 3 in. gauze pads so that they become 3 x 6 in., and lay the strips longitudinally on both sides of the incision. Place multiple layers of gauze along the opening.

8. Constantly use sterile PBS or physiological saline to keep the area of the incision and the exposed abdomen moist. The abdominal tissue and uterine membranes must not be allowed to dry out (see Note 35).

9. Use hemostats (for rats) or dog ear clips (for mice) to retract the skin and abdominal muscle wall. The two layers can be retracted together, or separately.

10. Mice and rats have both a right and left uterine horn, each comprised of several uterine swellings, that meet at the cervix (see Fig. 1). Gently remove the uterine horns from the abdominal cavity, and accurately count all the uterine swellings on each side (see Note 36). Dull forceps can be used to manipulate the uterine swellings. Make sure that all uterine swellings are accurately counted by identifying the rostral to the uppermost swelling, the ovary rostral to the uppermost swelling on each side. Diagram the arrangement of the uterine swellings and give each a number. By visual inspection, determine if all swellings contain a viable embryo (see Note 37).

11. Tuck the uterine swellings of one side back inside the abdominal cavity, and cover all the remaining uterine swellings with moist gauze to prevent them from becoming dry (see Note 38).

12. Thaw an aliquot of the retroviral supernatant. To every 50 pL of supernatant, add 2.5 pL of 1 mg/mL polybrene and 2.5 pL of 1% fast green dye; the final concentration of the polybrene and fast green dye is 0.5 mg/mL and 0.5%, respectively (see Note 39).

13. With careful positioning of the fiber optic light guides the uterine membranes are somewhat transparent. The embryo's body and head should be detectable. The embryonic sagittal and transverse dural sinuses demarcating the lateral ventricles should be recognizable. Using dull forceps, or some other comparable instrument, the position of the embryo and its head within the uterine swelling can be maneuvered (see Note 40). Use one motion, if possible, to penetrate the uterine membranes, skull and telencephalon (see Note 41). Once the pipet tip is in the lateral ventricle, the virus can be slowly released. The presence of the blue dye can be used to verify the placement of the pipet tip. The volume of the retrovirus to be injected will depend on the design of the experiment. Volumes of up to 5 pL can be safely injected.

14. After each embryo has been injected, replace all the uterine swellings back into the abdominal cavity. Suture closed the muscle and skin layers separately. First, close the inner muscle wall using silk suture thread; be sure that the fascial coverings of muscles do not slip into the abdominal cavity. Use interrupted sutures spaced about 14-3/8 in. apart. Similarly, suture closed the outer skin layer.

15. Place the pregnant mouse or rat in its cage under the infrared heat lamp. Cover half of the cage with aluminum foil to give the pregnant dam a choice of environmental temperature upon arousal and recovery; the tin foil will reflect the heat.

16. Discard all items that touched retrovirus in a dilute solution of bleach.

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