AAV9 Delivery of shRNA to the Mouse Heart

Hiroko Wakimoto1, J.G. Seidman1, Roger S.Y. Foo2, Jianming Jiang3

1 Department of Genetics, Harvard Medical School, Boston, Massachusetts, 2 Cardiovascular Research Institute (CVRI), Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 3 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 23.16
DOI:  10.1002/cpmb.9
Online Posting Date:  July, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


RNA interference (RNAi) is a rapid approach to dissect loss‐of‐function phenotype for a gene of interest. However, it is challenging to perform RNAi in specific organs and tissues in vivo. Engineered viruses can provide a useful tool for delivery of small RNAs in vivo. Recombinant adeno‐associated viruses (rAAVs) are the preferred method for delivering genes or gene modulators to target cells due to their high titer, low immune response, ability to transduce many types of cell, and overall safety. In this unit, we describe protocols for use of rAAVs as a cargo to deliver miRNA backbone‐based shRNA controlled by a cardiac‐specific promoter into the mouse heart. © 2016 by John Wiley & Sons, Inc.

Keywords: AAV; cardiovascular; shRNA

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Determination of RNAi Efficiency and Construction of AAV‐shRNA
  • Basic Protocol 2: Production of AAVs
  • Basic Protocol 3: Purification of AAV by Iodixanol Gradient Ultracentrifugation
  • Basic Protocol 4: AAV Transduction In Vivo
  • Reagents and Solutions
  • Commentary
  • Literature Cited
PDF or HTML at Wiley Online Library


Basic Protocol 1: Determination of RNAi Efficiency and Construction of AAV‐shRNA

  • pCAGIG plasmid (Addgene plasmid no. 11159)
  • LB agarose plates containing 100 μg/ml ampicillin (unit 1.1; Elbing and Brent, )
  • AAV vectors (available from Dr. Jiang Jianming):
    • pCAG‐cherry‐miR155
    • pAAV‐EGFP‐miR155
  • Competent cells:
    • Top10 (Thermo Fisher Scientific, cat. no. C4040‐10)
    • Stbl3 (Thermo Fisher Scientific, cat. no. C7373‐03)
  • LB medium containing 100 μg/ml ampicillin (unit 1.1; Elbing and Brent, )
  • QIAGEN Plasmid Miniprep Kit
  • Cloning primers for gene of interest
  • Restriction enzymes appropriate for cloning primers
  • BsmBI restriction enzyme (New England Biolabs)
  • shRNA primers (BLOCK‐iT miR RNAi; Thermo Fisher Scientific)
  • HEK293T cells (ATCC)
  • Complete DMEM (see recipe)
  • Sterile pipet tips
  • 30°C and 37°C shaking incubator
  • Nanodrop spectrophotometer
  • 6‐well culture plate
  • Fluorescence microscope
  • Additional reagents and equipment for PCR (unit 15.1; Kramer and Coen, ), DNA digestion (unit 3.1; Bloch, ), DNA ligation (unit 15.3; Mueller et al., 2002), RNA extraction (unit 4.1; Gilman, ), and real‐time PCR (unit 15.8; Bookout et al., )

Basic Protocol 2: Production of AAVs

  • pAAV2/9 (available from University of Pennsylvania Penn Vector Core)
  • pAdDeltaF6 (available from University of Pennsylvania Penn Vector Core)
  • QIAGEN Plasmid Maxi Kit
  • HEK293T cells (ATCC)
  • Transfection reagent (polyethylenimine [PEI]; e.g., Polysciences)
  • pAAV‐EGFP‐miR155 (available from Dr. Jiang Jianming)
  • Complete DMEM (see recipe)
  • 15‐cm culture plates
  • Centrifuge

Basic Protocol 3: Purification of AAV by Iodixanol Gradient Ultracentrifugation

  • HEK293T cells containing AAV (see protocol 2)
  • Lysis buffer (see recipe)
  • Dry ice/ethanol bath
  • Magnesium chloride
  • Benzonase Nuclease (e.g., Sigma)
  • 17%, 25%, 40%, and 60% (w/v) iodixanol (see recipe)
  • PBS ( appendix 22)
  • Vortex
  • 37°C water bath
  • Dounce tissue grinders (e.g., Wheaton)
  • Centrifuge (e.g., Beckman)
  • Beckman OptiSeal Polypropylene Tube, 36.2‐ml, 25 mm × 87 mm
  • 20‐cm blunt‐end needle
  • Ultracentrifuge (e.g., Beckman)
  • Ultracentrifuge rotor (e.g., Beckman VTi 50)
  • 19‐G needles
  • 3‐ml and 10‐ml syringes
  • Amicon Ultra‐15 Centrifugal Filter Units (nominal molecular weight limit of 100 kD)
  • 50‐ml Falcon tubes
  • 1.5‐ml microcentrifuge tube

Basic Protocol 4: AAV Transduction In Vivo

  • Mouse with newborn pups
  • Purified AAV (see protocol 3)
  • 3M Micropore Medical Tape
  • BD insulin syringe equipped with BD Ultra‐Fine Needle, 31‐G × 5/16" (0.3‐ml × 8 mm)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals.
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Bloch, K.D. 2001. Mapping by multiple endonuclease digestion. Curr. Protoc. Mol. Biol. 13:3.2.1‐3.2.5. doi: 10.1002/0471142727.mb0302s13.
  Bookout, A.L., Cummins, C.L., Mangelsdorf, D.J., Pesola, J.M., and Kramer, M.F. 2006. High‐throughput real‐time quantitative reverse transcription PCR. Curr. Protoc. Mol. Biol. 73:15.8.1‐15.8.28. doi: 10.1002/0471142727.mb1508s73.
  Bryant, L.M., Christopher, D.M., Giles, A.R., Hinderer, C., Rodriguez, J.L., Smith, J.B., Traxler, E.A., Tycko, J., Wojno, A.P., and Wilson, J.M. 2013. Lessons learned from the clinical development and market authorization of Glybera. Hum. Gene Ther. Clin. Dev. 24:55‐64. doi: 10.1089/humc.2013.087.
  Coumoul, X. and Deng, C.X. 2006. RNAi in mice: A promising approach to decipher gene functions in vivo. Biochimie 88:637‐643. doi: 10.1016/j.biochi.2005.11.010.
  Dietzl, G., Chen, D., Schnorrer, F., Su, K.C., Barinova, Y., Fellner, M., Gasser, B., Kinsey, K., Oppel, S., Scheiblauer, S., Couto, A., Marra, V., Keleman, K., and Dickson, B.J. 2007. A genome‐wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448:151‐156. doi: 10.1038/nature05954.
  Ding, J., Chen, J., Wang, Y., Kataoka, M., Ma, L., Zhou, P., Hu, X., Lin, Z., Nie, M., Deng, Z.L., Pu, W.T., and Wang, D.Z. 2015. Trbp regulates heart function through microRNA‐mediated Sox6 repression. Nat. Genet. 47:776‐783. doi: 10.1038/ng.3324.
  Elbing, K. and Brent, R. 2002. Media preparation and bacteriological tools. Curr. Protoc. Mol. Biol. 59:1.1.1‐1.1.7. doi: 10.1002/0471142727.mb0101s59.
  Gilman, M. 2002. Preparation of cytoplasmic RNA from tissue culture cells. Curr. Protoc. Mol. Biol. 58:4.1.1‐4.1.5. doi: 10.1002/0471142727.mb0401s58.
  Godbey, W.T., Wu, K.K., and Mikos, A.G. 1999. Poly(ethylenimine) and its role in gene delivery. J. Control. Release 60:149‐160. doi: 10.1016/S0168‐3659(99)00090‐5.
  Jiang, J., Wakimoto, H., Seidman, J.G., and Seidman, C.E. 2013. Allele‐specific silencing of mutant Myh6 transcripts in mice suppresses hypertrophic cardiomyopathy. Science 342:111‐114. doi: 10.1126/science.1236921.
  Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol. 56:15.1.1‐15.1.14. doi: 10.1002/0471142727.mb1501s56.
  Kamath, R.S., Fraser, A.G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., Welchman, D.P., Zipperlen, P., and Ahringer, J. 2003. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421:231‐237. doi: 10.1038/nature01278.
  Kunath, T., Gish, G., Lickert, H., Jones, N., Pawson, T., and Rossant, J. 2003. Transgenic RNA interference in ES cell‐derived embryos recapitulates a genetic null phenotype. Nat. Biotechnol. 21:559‐561. doi: 10.1038/nbt813.
  Lin, Z., Zhou, P., von Gise, A., Gu, F., Ma, Q., Chen, J., Guo, H., van Gorp, P.R., Wang, D.Z., and Pu, W.T. 2015. Pi3kcb links Hippo‐YAP and PI3K‐AKT signaling pathways to promote cardiomyocyte proliferation and survival. Circ. Res. 116:35‐45. doi: 10.1161/CIRCRESAHA.115.304457.
  Lin, Z., von Gise, A., Zhou, P., Gu, F., Ma, Q., Jiang, J., Yau, A.L., Buck, J.N., Gouin, K.A., van Gorp, P.R., Zhou, B., Chen, J., Seidman, J.G., Wang, D.Z., and Pu, W.T. 2014. Cardiac‐specific YAP activation improves cardiac function and survival in an experimental murine MI model. Circ. Res. 115:354‐363. doi: 10.1161/CIRCRESAHA.115.303632.
  Luo, J., Emanuele, M.J., Li, D., Creighton, C.J., Schlabach, M.R., Westbrook, T.F., Wong, K.K., and Elledge, S.J. 2009. A genome‐wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell 137:835‐848. doi: 10.1016/j.cell.2009.05.006.
  Mueller, P.R., Wold, B., and Garrity, P.A. 2001. Ligation‐mediated PCR for genomic sequencing and footprinting. Curr. Protoc. Mol. Biol. 56:15.3.1‐15.3.26. doi: 10.1002/0471142727.mb1503s56.
  Prendiville, T.W., Guo, H., Lin, Z., Zhou, P., Stevens, S.M., He, A., VanDusen, N., Chen, J., Zhong, L., Wang, D.Z., Gao, G., and Pu, W.T. 2015. Novel roles of GATA4/6 in the postnatal heart identified through temporally controlled, cardiomyocyte‐specific gene inactivation by adeno‐associated virus delivery of Cre recombinase. PLoS One 10:e0128105. doi: 10.1371/journal.pone.0128105.
  Vandenberghe, L.H., Wilson, J.M., and Gao, G. 2009. Tailoring the AAV vector capsid for gene therapy. Gene Ther. 16:311‐319. doi: 10.1038/gt.2008.170.
  Xiong, W., MacColl Garfinkel, A.E., Li, Y., Benowitz, L.I., and Cepko, C.L. 2015. NRF2 promotes neuronal survival in neurodegeneration and acute nerve damage. J. Clin. Invest. 125:1433‐1445. doi: 10.1172/JCI79735.
  Zamore, P.D. 2001. RNA interference: Listening to the sound of silence. Nat. Struct. Biol. 8:746‐750. doi: 10.1038/nsb0901‐746.
PDF or HTML at Wiley Online Library