Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Summary

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

Abstract

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.

Introduction

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 editing^2-5. A more routine laboratory method for gene manipulation has focused on the introduction of genetic materials (DNAs and RNAs containing coding sequences ....

Protocol

All described steps were performed under protocols approved by the Biosafety Committee and the Institutional Animal Care and Use Committee of Boston Children's Hospital. Boston Children'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 Assurance number: A3303-01). Mice were euthanized by CO₂ delivered from a compressed gas source. Tissue samples were collected after confirm....

Results

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)

Discussion

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 °C) with a lower shaking speed (180-200 rpm) c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Polyethylenimine, Linear (MW 25,000)	Polysciences, Inc.	#23966-2
Tube, Polypropylene, 36.2 ml, 25 x 87 mm, (qty. 56)	Beckman Coulter, Inc	# 362183
Nuclease, ultrapure	SIGMA	#E8263-25KU
Density Gradient Medium(Iodixanol)	SIGMA	#D1556-250ML
Centrifugal Filter Unit with Ultracel-100 membrane	EMD Millipore Corporation	#UFC910008
Laboratory pipetting needle with 90° blunt ends,gauge 14, L 6 in., nickel plated hub	SIGMA	#CAD7942-12EA
Poloxamer 188 solution (Pluronic® F-68 solution)	SIGMA	P5556-100ML
Proteinase K	SIGMA	3115828001
DNase I	Roche	10104159001
Centrifuge machine	Thermo Scientific	75004260
Centrifuge System	Beckman Coulter	363118
Ultracentrifuge	Beckman Coulter
DMEM medium	Fisher Scientific	SH30243FS
Fetal Bovine Serum	Atlanta Biologicals	S11150
rAAV9 vector	Penn Vector Core	P1967