Viral Gene Delivery in the Mouse Heart for Evaluating Gene Effects on Heart Function

Abstract

Source: Lu, A., et al. Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk. J. Vis. Exp. (2022)

The video demonstrates a method for ultrasound-guided intramyocardial injection of a recombinant viral vector in a mouse model. This technique enables targeted gene delivery to the heart for studying the effects of transgene expression on cardiac function.

Protocol

1. Viral transgene expression in rodent ventricular tissues

NOTE: Store recombinant adenovirus or adeno-associated virus (AAV) that expresses the target gene and the corresponding control virus, such as Ad-GFP (Green fluorescent protein) (titer of 1 x 10¹⁰ plaque-forming units [PFU]/mL) or AAV9-GFP (titer of 1 x 10¹³ genome copies [GC]/mL) (see Table of Materials), in a −80 °C freezer.

1. On the day of virus injection, thaw the virus on ice. Aspirate the virus expressing the target gene and the control virus into two separate syringes (volume = 50 µL) with 30 G ½ needles and keep on ice until use.
   NOTE: Any bubbles in the syringe and needle must be carefully removed.
2. Administer buprenorphine to rats or mice (0.05 mg/kg for rats, 0.1 mg/kg for mice, subcutaneous), one hour later, induce anesthesia using 3% isoflurane, and maintain isoflurane at 2% (in 100% oxygen at a flow rate of 0.5-1.0 L/min) for the subsequent procedure.
3. Gently pinch the animal's body (e.g., the tail) with a pair of forceps, and if the animal does not respond to the pinch with any body movements, proper anesthetization is confirmed.
4. Maintain body temperature at 37 °C using an electric heating pad. Shave the hair in the chest region using a hair clipper.
   NOTE: Care should be taken while using an electric heating pad due to potential uneven heat distribution.
5. Apply ophthalmic ointment to both eyes to prevent the drying of the cornea.
6. Keep the animal in a supine position and use a preclinical imaging system (see Table of Materials) for ultrasound imaging of the animal's heart in the short-axis orientation.
   NOTE: The following steps are performed in a level II biosafety cabinet which provides a sterile environment. Standard safety procedures, including those with safe needle handling, are employed.
7. Sterilize the left lower chest region of the animal with alternating rounds of an iodine-based or chlorhexidine-based scrub and alcohol three times in a circular motion.
8. Under ultrasound imaging guidance, insert the 30 G 1/2 needle of the syringe containing the virus as prepared in step 1.1. into the animal's chest via the left lower chest side of the body.
9. Approach the needle tip into the left ventricular front free wall and slowly inject 10-15 μL of the virus. Verify successful injection in ultrasound images by the enhanced brightness near the tip of the needle.
   NOTE: The amount of virus injected at each site is no more than 15 μL to prevent physical damage to the myocardial tissues.
10. Withdraw the needle from the heart and insert it into other regions of the left ventricle for a second and third injection of the same amount of virus.
    NOTE: The total number of injection sites is determined by the experimental design. If focal transgene expression is required, only one injection is needed; if more diffusive transgene expression is required, multiple injection sites (three to five) are usually needed.
11. After the completion of the injections, return the animal to its cage.
12. Carefully monitor the animal until it has regained sufficient consciousness to maintain sternal recumbency and return it to its housing room.
13. Administer buprenorphine hydrochloride (0.1 mg/kg, twice a day for mice, 0.05 mg/kg, twice a day for rats, subcutaneous) to the animal for the following 2 days. Return animals to the vivarium and monitor daily for any unusual signs of pain or distress, and if found, treat animals per the Institutional animal care committee protocols.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
30 G ½ PrecisionGlide Needle	Becton Dickinson (BD)	305106
Adeno-associated virus (Green flourescent protein(GFP) expressing AAV9)	Vector Biolabs	7007
Adenovirus (Ad-GFP)	Vector Biolabs	1060
Biosafety cabinet (Level II)	Microzone Corporation	N/A	Model #: BK-2-4
Buprenorphine	Vetergesic	DIN 02342510
Hair clipper	WAHL Clipper Corporation	78001
Hamilton syringe	Sigma-Aldrich	20701	705 LT, volume 50 μL
Heating pad	Life Brand	E12107
Isoflurane	Fresenius Kabi Ltd.	M60303
Mice (Ctnnb1flox/flox)	Jackson Labs	4152
Mice (αMHC-MerCreMer)	Jackson Labs	5650
Ophthalmic ointment	Systane	DIN 02444062
Rat (Sprague-Dawley, male)	Charles River	400
Scalpel blades	Fine Science Tools	10010-00
Scalpel handle	Fine Science Tools	10007-12
VEVO3100 Preclinical Imaging System	VisualSonic Inc.	N/A