In Utero Viral Transduction in a Mouse

Abstract

Source: Lutz, D., et al. Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord. J. Vis. Exp. (2019).

This video demonstrates viral transduction via in-utero injection in mouse embryos. A pregnant mouse is prepared for the surgery, and a midline incision is made to expose the uterine horns. A mixture of a virus containing the desired gene and a tracking dye is injected into the embryos within the uterine sacs at the targeted locations. Uterine horns are then placed back before suturing.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. In Utero Transduction

1. Prepare adeno-associated virus type 1 (AAV1) coding for the desired target (4 x 10¹¹ viral particles/µL of AAV1) in phosphate-buffered saline (PBS) at pH 7.4. Add 0.1 mg/µL Fast Green and keep the AAV1-Fast-Green mixture at 37 °C.
2. Prepare a thin capillary tip with the desired shape (8 mm in length, with an outside diameter of 80 µm and an inside diameter of 50 µm) using a micropipette puller (settings: pressure = 500, heat = 700, pull = 0, velocity = 80, time = 200, see the Table of Materials). Break the tip of the capillary so that it is 4-5 mm.
3. Assemble an aspirator tube (44 cm x 0.7 cm) with the capillary tip and aspirate 15 µL of the AAV1-Fast-Green mixture into the capillary.
4. Keep the animal subjects at a constant physiological body temperature of 37 °C throughout the entire procedure.
5. Place a pregnant C57Bl/6 mouse (embryonic day 14.5) into the preincubation chamber and anesthetize the mouse with gaseous 4% isoflurane (with a volumetric airflow rate of 0.6-0.8 L/min).
6. Subcutaneously, inject buprenorphine (0.1 mg/kg of body weight).
7. Place the anesthetized mouse on the prewarmed surgical plate (37 °C).
8. Cover the eyes with a lubricant.
9. Fit the mouse with the anesthesia mask (gaseous 1.5 % isoflurane at a volumetric airflow rate of 0.6-0.8 L/min) on the surgical plate and shave the abdominal skin region. Wipe the shaved region with 3X 75% ethanol and then with betadine solution.
   NOTE: Monitor the breathing behavior of the anesthetized mouse continuously. Adjust the concentration of the isoflurane gas according to the inhalation-exhalation pattern of the mouse.
10. Check for the absence of the plantar reflex by squeezing the hind paw phalanges of the mouse.
11. Open the abdominal cavity by gripping the skin with curved serrated iris forceps (10 cm) and cutting the skin along the linea mediana with straight tungsten carbide scissors (10 cm), and then, by gripping the peritoneal wall with straight Dumont tweezers (12 cm, 0.2 mm x 0.12 mm) and cutting the wall along the linea alba with straight Vanna's scissors (8 cm).
12. Place a piece of fenestrated paraffin film on the abdominal opening and fix the film on both ends with micro-mosquito hemostatic forceps (12.5 cm, curved).
13. Expose the uterine horns with a spoon-like device to avoid damage to the embryos inside the uterine horns. Drip a few drops of PBS (37 °C) on the uterine horns and inspect the embryos for damage or malformations inside the uterine sac.
14. Document the order and position of the embryos in the uterine horns. Turn the embryos carefully inside the uterine sac until the desired position for injection is reached.
15. Inject 1-2 µL of the AAV1-Fast-Green mixture by visually inspecting the injection site (e.g., brain ventricles) and the dye penetration under a stereomicroscope.
16. Document the injected embryos and place the uterine horns with the injected embryos back into the abdominal cavity.
17. Drip a few drops of PBS (37 °C) into the abdominal cavity. Close the cavity by suturing the peritoneal wall (use polyamide 6-0-sized sutures) and the skin (use polyamide 3-0-sized sutures) using Halsted's mosquito hemostatic forceps (12.5 cm, curved). Alternatively, use a simple interrupted pattern or stainless-steel suture/staples to close the abdominal cavity and the skin.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
26Gx 1'' needle	Henke-Sass, Wolf GmbH
410 Anaesthesia Unit for air pump	Biomedical Instruments (Univentor)	8323102
Adeno-associated virus serotype 1 (AAV1)	UKE (Viral Core Facility)	-	For references and target areas of AAV1 see: https://www.addgene.org/viral-vectors/aav/aav-guide/ and also: Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer. Kwon I, Schaffer DV. Pharm Res. 2008 Mar;25(3):489-99. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P, Zolotukhin S, Reier PJ, Mandel RJ, Muzyczka N. Mol. Ther. 2004 Aug;10(2):302-17. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. McCarty DM, Monahan PE, Samulski RJ. Gene Ther. 2001 Aug;8(16):1248-54.
Air Pump	Biomedical Instruments (Univentor)	Eheim 100
Aspirator tune assemblies	Sigma-Aldrich	A5177-5EA
Breathing Mask Mouse Anodized Aluminium	Biomedical Instruments (Univentor)	-
Buprenorphine	Temgesic	ampules	painkiller
Capillaries	Science-Products	GB100TF-10	with fillament
Dumont tweezers (#3, 12 cm, straight, 0.2 x 0.12 mm)	FST	11203-23
Electric shaver	Phillips	-
Ethicon sutures (Ethilon, 6-0 and 3-0)	Ethicon	-	polyamide
Eye lubricant	Bepanthene	-
Fast Green	Sigma-Aldrich	F7252	for visualization of injected liquids
Gas Routing Switch 4/2 connectors	Biomedical Instruments (Univentor)	8433020
Halsted Mosquito hemostatic forceps (12.5 cm, straight)	FST	13011-12
Induction box for micewith horizontally moving lid.Inner dimensions: LxBxH: 155x115x130 mm.Wall thickness: 6 mm	Biomedical Instruments (Univentor)	-
Iris forceps (10cm, curved, serrated)	FST	14007-14
Iris scissors (11cm, straight, tungsten carbide)	FST	14501-14
Isofluran OP Tisch, electrically heated, smOuter dimensions: 257x110x18 mm.Heating area: 190x90 mmThe removal of the isoflurane escapingthe breathing mask is downwards in compliance with theregulations	Biomedical Instruments (Univentor)	-
Isoflurane (Attane)	JD medical		inhalation anesthesia
Light microscope Basic DM E	Leica	-	4x (N.A. 0.1 ∞/-), 10x (N.A. 0.22 ∞/0.17), 40x (N.A. 0.65 ∞/0.17), 100x (N.A. 1.25 ∞/0.17) objectives
Micropipette pulle	Science-Products	P-97
Mosquito hemostatic forceps (12.5cm, curved)	FST	13010-12
Thermopuller P-97	Sutter Instruments
Tygon 6/4 Tubing material for connection of all partsOuter diameter: 6mmInner diameter: 4mmWall thickness: 1mm	Biomedical Instruments (Univentor)
Vannas scissors (8 cm, straight)	FST	15009-08