<p class='jove_title'><strong>1. Treat neonatal mice with rAAV9 at P0.5-P2.5 by subcutaneous injection.</strong></p>
<ol>
	<li>Pre-fill a 29G1/2, 0.33 x 12.7 mm insulin syringe with the recombinant adeno-associated virus 9 (rAAV9) solution. Be careful to remove air bubbles.</li>
	<li>Hold the&#160;cryo-anesthetized pup in one hand with thumb and forefinger. Prior to injection, swipe the back skin of the pup with a swab stick saturated with 70% isopropyl alcohol to maintain the sterile condition. Insert the syringe needle into the anterior-dorsal subcutis of the animal at an angle of 5 to 10&#176;. Inject 50-70 &#181;l of the rAAV9 solution using the insulin syringe.&#160;&#160;&#160;&#160;<br />
	<strong>NOTE</strong>: rAAV9 can also be delivered to the mouse via intraperitoneal or intravenous injection. Efficient expression of the delivered genes in the heart can be obtained. However, intraperitoneal injection sometimes may result in leaky expression in the liver. After injection, the condition of the pups was monitored every day.</li>
</ol>

<table><tbody><tr><td>rAAV9 vector&amp;nbsp;</td><td>Penn Vector Core&amp;nbsp;</td><td>P1967</td><td></td></tr></tbody></table>

recombinant adeno associated virus based gene delivery: a technique for transgene delivery and cardiac-specific expression in rodent model

Recombinant Adeno Associated Virus Based Gene Delivery: A Technique for Transgene Delivery and Cardiac-Specific Expression in Rodent Model

<p class='jove_content'>Source: Ding, J. <em>et al</em>. <a href='https://www.jove.com/v/54787'>Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts.</a> <em>J. Vis. Exp.</em> (2016)</p>
<p class='jove_content'>In this video, we demonstrate recombinant adeno associated virus-based gene delivery in the mouse pup. This method helps in studying gene expression in the targeted cells.</p>

Source: Ding, J. et al. Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts. J. Vis. Exp. (2016)
In this video, we demonstrate ...

<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>

<p>After determining the viral titer according to the text protocol, prepare rAAV9 working solutions with virus titers of 1-7 x 10<sup>12</sup> 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.</p>
<p>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.</p>

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2.9K Views. 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 he...

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<table><tbody><tr><td>rAAV9 vector&nbsp;</td><td>Penn Vector Core&nbsp;</td><td>P1967</td><td></td></tr></tbody></table>

Recombinant Adeno Associated Virus Based Gene Delivery: A Technique for Transgene Delivery and Cardiac-Specific Expression in Rodent Model

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