Injecting Recombinant Adeno-Associated Vectors in a Spinal and Bulbar Muscular Atrophy Mouse Model

Abstract

Source:
Pourshafie, N., et al. Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents. J. Vis. Exp. (2018).

In this video, a spinal and bulbar muscular atrophy or SBMA-affected mouse undergoes tail vein injection of recombinant AAV (Adeno-associated vectors) vectors carrying a plasmid encoding therapeutic microRNA. The vector produces microRNA that binds to mutant AR (Androgen receptor)-mRNA (messenger ribonucleic acid), triggering its degradation. This reduces toxic protein accumulation and helps to improve neuromuscular function.

Protocol

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

1. Tail Vein Injection of AAV-miRNA Plasmid

NOTE: This step needs adjustment according to the target gene and the age and weight of the mice. Use the Institutional and Animal Care and Use Committee (IACUC) guidelines to determine the dose range, volume and best route of administration based on the age and weight of the mice. GFP (Green fluorescent protein) fluorescence signal and miRNA expression levels in target tissues are expected to peak starting 2 weeks after the injection. The following protocol refers to male mice in early adulthood. AAV should be handled as a biohazard under Biosafety Level 1 guidelines.

1. Divide AAV-miRNA plasmid stock in 100-200 µL aliquots containing a viral load of 10¹⁰-10¹¹ viral genomes per mL (vg/mL) using sterile phosphate buffered saline (PBS). Laboratory biohazard personal protective equipment should be used to handle the AAV solution.
   NOTE: Once an aliquot is thawed it can be stored at 4 °C for up to two weeks. Viral stock can be stored in -80 °C freezer.
2. Restrain the mouse in commercially available restrainers such as plastic or tapered plastic film tube of proper size.
3. Clean the surface of the tail with 70% alcohol wipes.
4. Hold a warm pad (28-30 °C) under the tail for 30 seconds to increase vasodilation. Alternatively, warm the animals using a red heat lamp (at a distance of 2-3 feet) or by dipping the tail into warm water.
5. Starting from the distal portion of the tail, insert the needle (27-30 G) loaded with the viral aliquot, bevel up, 15° from the tail, into the vein. Before injection, ensure the needle is inserted correctly.
   1. If resistance is felt during injection or swelling appears under the skin, stop the procedure. Remove the needle and re-insert it above the previous injection site.
      NOTE: Alternatively, this can be achieved by gently aspirating the needle and observing blood. Hard aspiration can collapse the tail vein. If the vein blanches during injection, the needle is inserted correctly.
6. After the injection has been completed, wait a few seconds before raising the needle.
7. Observe the injection site for bleeding. Apply moderate pressure using two fingers to the site until bleeding stops.
8. Return the animal to its cage.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Scale	Pelouze	SP5
Nalgene bucket	Thermo Scientific	12014000	4000 mL
Microcentrifuge tube	USA Scientific	1615-5500
Lactated Ringer's Solution	Baxter Healthcare	0338-0114-04
Mini vortexer	Fisher Scientific	128101
Centrifuge	Eppendorf	22624415
Laboratory film	Bemis	PM996
1-mL syringe	BD	309626	Comes with a 25g needle attached. Replace with 27-30g needle for injection
30g needle	BD	305106
Isoflurane	Piramal Healthcare	66794-013-25	Isoflurane USP 250 mL
Anesthesia mask	Kopf Instruments	906
Gauze	Henry Schein	100-2524	2" x 2" (5cm x 5cm)
Pipet tips	USA Scientific	1111-0816
Micropipet	Gilson	4642080
Gas anesthesia vaporizer	SurgiVet	4214322	Classic T3
Gas anesthesia scavenger	SurgiVet	32373B10	Enviro-PURE
Heating pad	Sears	17280
Bench pad	VWR	56617-006
Rotarod	Panlab/Harvard Apparatus	76-0772	4 rats/ 4 mice
70% Ethanol	Decon Laboratories, Inc.	2401	140 proof
70% Isopropanol	VWR	89108-160