Name: preparation of raav9 to overexpress or knockdown genes in mouse hearts
Uploaded: 2016-12-17T13:00:00
Description: Watch this Scientific Journal Video about Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts at JoVE.com

We thank Dr. Zaffar Haque for careful reading of the manuscript. We thank Drs. Masaharu Kataoka and Gengze Wu for discussions and help. Work in the Wang lab is supported by the American Heart Association, Muscular Dystrophy Association, and NIH (HL085635, HL116919, HL125925).

All described steps were performed under protocols approved by the Biosafety Committee and the Institutional Animal Care and Use Committee of Boston Children&#39;s Hospital. Boston Children&#39;s Hospital has pathogen-free mouse facilities with regulated light/dark cycles and climate control. Veterinary and animal care staff change cages and ensure the health of the mice. The facilities are AAALAC certified and have active Animal Welfare Assurance certification (AAALAC Accreditation Granted on 2/24/1992. Animal Welfare A...

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It is important to minimize undesired ITR recombination during plasmid construction. Before generating the virus, one must always monitor the ITR integrity of the AAV plasmids by using restriction digestion and agarose gel electrophoresis. It is impossible to obtain 100% intact plasmids, but the recombination ratio should be minimized as much as possible. Less than 20% is acceptable for successful rAAV9 packaging. Of note, culturing the bacteria at lower temperature (30 &#176;C) with a lower shaking speed (180-200 rpm) c...

A correction was made to the Authors section in: <a href="http://www.jove.com/video/54787">Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts</a>.
One of the authors names and affiliation was corrected from:
Jian-Ming Jiang3,4 
3 Department of Genetics, Harvard Medical School 
4 Howard Hughes Medical Institute
to:
Jianming Jiang5 
5 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore

A correction was made to the Authors section in: <a href="http://www.jove.com/video/54787">Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts</a>.

Erratum: Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling expression or activity of specific genes in various biological systems has become a valuable strategy in the study of gene function1. A direct means of accomplishing this goal is to manipulate nucleotide sequences and generate mutant alleles. Although making precise, targeted changes to the genome of living cells is still a time-consuming and labor-intensive practice, the development of the powerful TALEN and Crispr/Cas9 tools has opened a new era of genome editing2-5. A more routine laboratory method for gene manipulation has focused on the introduction of genetic materials (DNAs and RNAs containing coding sequences or siRNAs/shRNAs) into the cells to express or knockdown the gene(s) of interest1,6.
In many cases, the major bottleneck for gene manipulation is the delivery of DNA, RNA, or protein into the cells. With regard to in vitro studies, efficient transfection systems have been established in many cultured cell lines. However, in the mouse model in particular, in vivo gene delivery is more challenging. There are a series of extra- and intracellular barriers that need to be bypassed in order to achieve efficient cellular uptake of the exogenous reagents. Additional obstacles include the rapid clearance and the short duration of the delivered materials7,8. One strategy to circumvent these issues is to use viral vectors as &#34;carriers&#34; or &#34;vehicles&#34; for in vivo gene delivery. The naturally-evolved transduction properties of viruses allow the efficient delivery of a gene of interest into cells7,9,10. Numerous types of viral vectors have been developed and enable flexible in vivo gene manipulation in different cell types and organs in mice.
The most commonly-used viral systems include Retrovirus, Lentivirus, Adenovirus, and Adeno-associated virus (AAV)11. Retroviruses are single-stranded RNA viruses and can introduce their genetic material to the host cell genome in a stable manner during mitotic division, providing the potential for lifelong expression of the transduced genes in the target cells and organs12-14. However, many types of retroviruses only infect dividing cells, and their efficacy in non-dividing cells is very low15. This limits their utility for gene delivery. Lentivirus is a genus of the Retroviridae family. Different from other retroviruses, Lentivirus can infect both dividing and non-dividing cells and has been widely used for gene transfer into post-mitotic and highly-differentiated cells16. The life cycle of Lentivirus also involves the integration of vector DNA into the host genome. Thus, Lentivirus-mediated gene delivery enables stable and long-duration expression of the transduced genetic elements16-18. However, this feature may represent a double-edged sword in the use of these viruses to manipulate gene expression, as integration of vector DNA may lead to insertional mutagenesis in the host cells and can cause artefactual effects. Adenovirus is another widely-used gene delivery system. Unlike retroviruses and lentiviruses, Adenoviruses are non-integrated and do not interfere with the genomic integrity of host cells8,10,11,19. In addition, Adenoviruses can transfect DNA into many cell types, and infection is not dependent upon active cell division19. Another important characteristic of Adenoviruses is the ease of vector purification, as the viral vectors have the ability to be replicated19,20. However, the major caveat of this system is that Adenovirus infection can trigger strong immune responses in target cells and organs19, restricting its use in many investigations, particularly in gene therapy studies.
Compared with these different types of viral vectors, recombinant Adeno-associated virus (rAAV) appears to be the ideal gene delivery system21,22. It exhibits minimal immunogenicity and pathogenicity23,24. In addition, rAAV infects a broad range of cell types, including both dividing and non-dividing cells. In most cases, rAAV does not integrate into host genomes; thus, the risk of undesired genetic or genomic changes in the target cells is low22.
Recently, rAAV systems have been successfully used for the in vivo delivery of DNA encoding proteins, miRNAs, shRNAs, and Crispr-gRNAs into mouse cardiac muscle23,25-29. This methodology has facilitated fundamental investigations and gene therapy studies in the field of cardiovascular research. Here, the detailed procedure to generate rAAV9 vectors that efficiently overexpress or knockdown the genes of interest in mouse hearts was described. The protocol provides a simple and effective method of manipulating cardiac gene expression in murine experimental models.

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			<td height="21" style="height:21px;width:575px;">Polyethylenimine, Linear (MW 25,000)</td>
			<td style="width:241px;">Polysciences, Inc.&#160;</td>
			<td style="width:192px;">#23966-2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Tube, Polypropylene, 36.2 mL, 25 x 87 mm, (qty. 56)</td>
			<td style="width:241px;">Beckman Coulter, Inc</td>
			<td style="width:192px;"># 362183</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Nuclease, ultrapure</td>
			<td style="width:241px;">SIGMA</td>
			<td style="width:192px;">#E8263-25KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Density Gradient Medium(Iodixanol)</td>
			<td style="width:241px;">SIGMA</td>
			<td style="width:192px;">#D1556-250ML</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Centrifugal Filter Unit with Ultracel-100 membrane</td>
			<td style="width:241px;">EMD Millipore Corporation</td>
			<td style="width:192px;">#UFC910008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Laboratory pipetting needle with 90&#176; blunt ends,gauge 14, L 6 in., nickel plated hub</td>
			<td style="width:241px;">SIGMA</td>
			<td style="width:192px;">#CAD7942-12EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Poloxamer 188 solution (Pluronic&#174; F-68 solution)</td>
			<td style="width:241px;">SIGMA</td>
			<td style="width:192px;">P5556-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Proteinase K</td>
			<td style="width:241px;">SIGMA</td>
			<td style="width:192px;">3115828001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">DNase I</td>
			<td style="width:241px;">Roche</td>
			<td style="width:192px;">10104159001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Centrifuge machine</td>
			<td style="width:241px;">Thermo Scientific</td>
			<td style="width:192px;">75004260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Centrifuge System</td>
			<td style="width:241px;">Beckman Coulter</td>
			<td style="width:192px;">363118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Ultracentrifuge</td>
			<td style="width:241px;">Beckman Coulter</td>
			<td style="width:192px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">DMEM medium</td>
			<td style="width:241px;">Fisher Scientific</td>
			<td style="width:192px;">SH30243FS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Fetal Bovine Serum&#160;</td>
			<td style="width:241px;">Atlanta Biologicals&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:192px;">S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">rAAV9 vector</td>
			<td style="width:241px;">Penn Vector Core</td>
			<td style="width:192px;">P1967</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of raav9 to overexpress or knockdown genes in mouse hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

The strategies for rAAV9 construction of rAAV9.cTNT::GFP or rAAV9.U6::shRNA plasmids are shown in Figures 1 and 2, respectively. As the examples, the rAAV9 vector was generated to overexpress the GFP gene in mouse hearts. The resulting plasmid contains the cTNT::GFP cassette flanked by two ITR sites (Figure 1). The rAAV9.U6::shRNA vector was constructed to knockdown Trbp mRNA (Figure 2)
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Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

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