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Using injectoporation to deliver genes to mechanosensory hair cells

Nature Protocols volume 9, pages 2438–2449 (2014)Cite this article

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Abstract

Mechanosensation, the transduction of mechanical force into electrochemical signals, allows organisms to detect touch and sound, to register movement and gravity, and to sense changes in cell volume and shape. The hair cells of the mammalian inner ear are the mechanosensors for the detection of sound and head movement. The analysis of gene function in hair cells has been hampered by the lack of an efficient gene transfer method. Here we describe a method termed injectoporation that combines tissue microinjection with electroporation to express cDNAs and shRNAs in mouse cochlear hair cells. Injectoporation allows for gene transfer into dozens of hair cells, and it is compatible with the analysis of hair cell function using imaging approaches and electrophysiology. Tissue dissection and injectoporation can be carried out within a few hours, and the tissue can be cultured for days for subsequent functional analyses.

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Figure 1: Tissue dissection.
Figure 2: Injectoporation setup.
Figure 3: Diagram of the microinjection procedure and evaluation of the injectoporation efficiency.
Figure 4: Histological analysis of injectoporated hair cells.
Figure 5: Physiological analyses of mechanically activated Ca2+ responses and membrane currents.
Figure 6: Analysis of CDH23 functions in OHCs.

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Acknowledgements

We thank J. Bartles (Northwestern University) for espin-EGFP. This research was supported by funding from the US National Institutes of Health (NIH) (to U.M., DC005965 and DC007704), the Dorris Neuroscience Center (to U.M.), the Deutsche Forschungsgemeinschaft (to T.W.) and the Bundy foundation (to W.X.).

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Author notes

  1. Wei Xiong & Nicolas Grillet

    Present address: Present addresses: School of Life Sciences, Tsinghua University, Beijing, China (W.X.); Department of Otolaryngology, Stanford University, Stanford, California, USA (N.G.).,

Authors and Affiliations

  1. Department of Molecular and Cellular Neuroscience, The Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, California, USA

    Wei Xiong, Thomas Wagner, Linxuan Yan, Nicolas Grillet & Ulrich Müller

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  1. Wei Xiong
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  2. Thomas Wagner
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Contributions

W.X., T.W. and U.M. developed protocols and designed the devices; W.X., T.W., L.Y. and N.G. performed experiments; and W.X. and U.M. analyzed the data and wrote the manuscript.

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Correspondence to Wei Xiong or Ulrich Müller.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Gene transfer into supporting cells by injectoporation.

A diagram of a cross section through the organ of Corti is shown as well as the EGFP fluorescence signal in the indicated cell types. Scale bars: 10 μm.

Supplementary information

Supplementary Figure 1

Gene transfer into supporting cells by injectoporation. (PDF 467 kb)

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Xiong, W., Wagner, T., Yan, L. et al. Using injectoporation to deliver genes to mechanosensory hair cells. Nat Protoc 9, 2438–2449 (2014). https://doi.org/10.1038/nprot.2014.168

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