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Tol2-Mediated Delivery of miRNAs to the Chicken Otocyst Using Plasmid Electroporation

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Auditory and Vestibular Research

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1427))

Abstract

The avian embryo has a well-documented history as a model system for the study of neurogenesis, morphogenesis, and cell fate specification. This includes studies of the chicken inner ear that employ in ovo electroporation, in conjunction with the Tol2 system, to yield robust long-term transgene expression. Capitalizing on the success of this delivery method, we describe a modified version of the Tol2 expression vector that readily accepts the insertion of a microRNA-encoding artificial intron. This offers a strategy to investigate the possible roles of different candidate microRNAs in ear development by overexpression. Here, we describe the general design of this modified vector and the electroporation procedure. This approach is expected to facilitate phenotypic screening of candidate miRNAs to explore their bioactivity in vivo.

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Acknowledgements

We thank Ulrike Sienknecht for providing a protocol for electroporation electrode fabrication and Kaidi D. Zhang for converting pTol2-GFP into a Gateway® destination vector. We thank Vidhya Munnamalai for helping with figure preparation, Andrea Battisti for piloting electroporation techniques in our lab, and M. Katie Scott for reviewing the manuscript. Support from the NIDCD (R01DC002756 to D.M.F. and F31DC0011687 to M.L.S.) is gratefully acknowledged. Plasmid constructions were aided by data obtained from the DNA sequencing facility at Purdue University supported by P30 CA023168.

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Authors and Affiliations

  1. Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT, USA

    Michelle L. Stoller

  2. Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN, 47907-2054, USA

    Donna M. Fekete

  3. Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA

    Donna M. Fekete

Authors
  1. Michelle L. Stoller
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  2. Donna M. Fekete
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Correspondence to Donna M. Fekete .

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Editors and Affiliations

  1. College of Medicine, University of South Florida, Tampa, Florida, USA

    Bernd Sokolowski

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Stoller, M.L., Fekete, D.M. (2016). Tol2-Mediated Delivery of miRNAs to the Chicken Otocyst Using Plasmid Electroporation. In: Sokolowski, B. (eds) Auditory and Vestibular Research. Methods in Molecular Biology, vol 1427. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3615-1_2

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  • DOI: https://doi.org/10.1007/978-1-4939-3615-1_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3613-7

  • Online ISBN: 978-1-4939-3615-1

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