Injection of Retrovirus into Fetuses

Accurately establish the gestational age of the embryonic animals to be injected with retrovirus. The question to be addressed will dictate at which gestational age the retrovirus should be injected, and will be related to the developmental time course of the organ or structure under examination. The time point under analysis should coincide with the period of cell genesis for the structure under examination since the retroviral vectors only infect dividing cells.

Pregnant dams of a known gestational age can be obtained from a supplier. Alternatively, the investigator can establish a breeding colony and determine when insemination has occurred according to routinely used procedures. By convention, the day a vaginal plug is detected is designated embryonic d 0 (E0). Gestation usually lasts 20 d for mice and 22 d for rats.

1. A long-acting anesthetic, such as chloral hydrate should be used (see Note 6). The effective intraperitoneal dose of chloral hydrate is approx 400 mg/kg for rodents. Dissolve chloral hydrate in 0.1 M PBS or 0.9% NaCl. A 3-cc syringe and 25 gage needle are needed to administer the chloral hydrate.

2. Electric shaver.

3. A bifurcated fiber optic light source to transilluminate the uterine swellings (see Note 7).

4. Pneumatic picopump for controlled delivery of retrovirus. There are several pneumatic picopumps available from various vendors, including one manufactured by World Precision Instruments (Sarasota, FL) (Model # PV 820) that is suitable for injections of small volumes. The operation of a pneumatic picopump also requires a dedicated nitrogen gas tank and vacuum line.

5. Supplies for the laparoscopic surgery and intraventricular retroviral injections:

b. Sterile PBS.

d. Capillary tubing (Omega dot borosilicate glass tubing, # 30-30-1, FHC, ME).

6. Surgical platform and collecting tray for supporting the experimental animal above the reservoir of irrigation fluids during surgery. Any acrylic or metal tray large enough to accommodate the mouse or rat with extended limbs and that is raised in some fashion 1-3 cm above the base can serve as a platform. The designated platform should fit inside a tray, of slightly larger dimensions than the platform, so that the irrigation fluid (PBS) will not run all over the operating surface. A shallow metal tray will work well for this purpose.

7. Surgical instruments for laparoscopic surgery (see Note 8):

a. Small scissors.

b. 2 Hemostats (rat surgery only).

c. 2 Dog ear clips (mouse surgery only).

d. Small toothed forceps.

h. Needle holder.

i. #5 (rat) or # 6 (mouse) suture thread and needles (Ethicon Inc., Somerville, NJ).

8. Retroviral supernatant a. Ice bucket filled with ice.

b. 1% Fast green dye (F7252, Sigma, St. Louis, MO).

c. 1 mg/mL polybrene, prepare as in Subheading 2.1.2., item 3d.

d. Aliquot of frozen viral supernatant.

The viral supernatant, fast green dye, and polybrene should all be kept on ice when not in use.

9. An infrared heat lamp should be used to warm the pregnant dam while recovering from surgery (see Note 9). The infrared light source, with shield, can be bought at a hardware store, and attached on an adjustable stand, if desired.

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