This article discusses the use of recombinant adeno-associated virus (rAAV) for targeted gene delivery to cardiomyocytes in neonatal mice. The methodology involves subcutaneous injection of the rAAV vector, which facilitates gene expression in the heart.
Recombinant adeno-associated virus, rAAV, is a small, single-stranded DNA virus genetically engineered to target a specific cell type and transport its genetic cargo to the host cell nucleus for gene expression.
Begin by restraining a cryo-anesthetized mouse pup. Sterilize the back skin and subcutaneously inject the rAAV vector carrying the desired transgene along a cardiac-specific promoter. The vector gets absorbed into the vascular system and reaches distant organs, including the heart.
Here, the recombinant virus attaches to the surface of the cardiomyocyte and gets internalized via endocytosis into an endosome. Within the endosome, the viral capsid partially disassembles, exposing the viral lytic proteins. This protein induces pores in the endosomal membrane, through which the vector escapes into the cytoplasm.
The vector travels across cytoplasmic microtubules and enters through the nuclear pore. Once in the nucleus, the viral capsid disassembles to release its genome. The single-stranded DNA carrying the target gene is then converted into double-stranded DNA.
Following DNA transcription, the produced mRNA moves into the cytoplasm. Here, the mRNA undergoes translation, expressing the target protein in the cardiomyocytes. Observe tissue samples from different organs under a fluorescence microscope to determine the efficiency and tissue specificity of the gene delivery to the heart.
After determining the viral titer according to the text protocol, prepare rAAV9 working solutions with virus titers of 1-7 x 1012 particles per milliliter. To treat neonatal mice, use the rAAV9 solution to prefill a 0.33 x 12.7-milliliter insulin syringe with a 29-gauge 1/2-inch needle, avoiding air bubbles.
After cryoanesthetizing a pup, hold the pup in one hand with thumb and forefinger. Then, swipe the back skin of the pup with a swab stick saturated with 70% isopropyl alcohol. Insert the syringe needle into the anterior-dorsal subcutis of the animal at an angle of 5 to 10 degrees before injecting 50 to 70 microliters of the rAAV9 solution.
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