Skip to main content
SpringerLink
Log in

Helios® Gene Gun-Mediated Transfection of the Inner Ear Sensory Epithelium: Recent Updates

  • Protocol
  • First Online:
Auditory and Vestibular Research

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

Abstract

The transfection of vertebrate inner ear hair cells has proven to be challenging. Therefore, many laboratories attempt to use and improve different transfection methods. Each method has its own advantages and disadvantages. A particular researcher’s skills in addition to available equipment and the type of experiment (in vivo or in vitro) likely determine the transfection method of choice. Biolistic delivery of exogenous DNA, mRNA, or siRNA, also known as Helios® Gene Gun-mediated transfection, uses the mechanical energy of compressed helium gas to bombard tissue with micron- or submicron-sized DNA or RNA-coated gold particles, which can penetrate and transfect cells in vitro or in vivo. Helios® Gene Gun-mediated transfection has several advantages: (1) it is simple enough to learn in a short time; (2) it is designed to overcome cell barriers even as tough as plant cell membrane or stratum corneum in the epidermis; (3) it can transfect cells deep inside a tissue such as specific neurons within a brain slice; (4) it can accommodate mRNA, siRNA, or DNA practically of any size to be delivered; and (5) it works well with various cell types including non-dividing, terminally differentiated cells that are difficult to transfect, such as neurons or mammalian inner ear sensory hair cells. The latter advantage is particularly important for inner ear research. The disadvantages of this method are: (1) low efficiency of transfection due to many variables that have to be adjusted and (2) potential mechanical damage of the tissue if the biolistic shot parameters are not optimal. This chapter provides a step-by-step protocol and critical evaluation of the Bio-Rad Helios® Gene Gun transfection method used to deliver green fluorescent protein (GFP)-tagged full-length cDNAs of myosin 15a, whirlin, β-actin, and Clic5 into rodent hair cells of the postnatal inner ear sensory epithelia in culture.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Xiong W, Wagner T, Yan L, Grillet N, Müller U (2014) Using injectoporation to deliver genes to mechanosensory hair cells. Nat Protoc 9:2438–2449

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Brigande JV, Gubbels SP, Woessner DW, Jungwirth JJ, Bresee CS (2009) Electroporation-mediated gene transfer to the developing mouse inner ear. Methods Mol Biol 493: 125–139

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Luebke AE, Foster PK, Muller CD, Peel AL (2001) Cochlear function and transgene expression in the guinea pig cochlea, using adenovirus- and adeno-associated virus-directed gene transfer. Hum Gene Ther 12:773–781

    Article  CAS  PubMed  Google Scholar 

  4. Konishi M, Kawamoto K, Izumikawa M, Kuriyama H, Yamashita T (2008) Gene transfer into guinea pig cochlea using adeno-associated virus vectors. J Gene Med 10:610–618

    Article  CAS  PubMed  Google Scholar 

  5. Akil O, Seal RP, Burke K, Wang C, Alemi A, During M, Edwards RH, Lustig LR (2012) Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy. Neuron 75:283–293

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Chien WW, Isgrig K, Roy S, Belyantseva IA, Drummond MC, May LA, Fitzgerald TS, Friedman TB, Cunningham LL (2016) Gene Therapy Restores Hair Cell Stereocilia Morphology in Inner Ears of Deaf Whirler Mice. Mol Ther 24(1):17–25

    Google Scholar 

  7. Askew Ch, Rochat C, Pan B, Asai Y, Ahmed H, Child E, Schneider BL, Aebischer P, Holt JR (2015) Tmc gene therapy restores auditory function in deaf mice. Sci Translat Med 7:295ra108

    Google Scholar 

  8. Woods C, Montcouquiol M, Kelley MW (2004) Math1 regulates development of the sensory epithelium in the mammalian cochlea. Nat Neurosci 7:1310–1318

    Article  CAS  PubMed  Google Scholar 

  9. Zheng JL, Gao WQ (2000) Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears. Nat Neurosci 3:580–586

    Article  CAS  PubMed  Google Scholar 

  10. Driver EC, Kelley MW (2010) Transfection of mouse cochlear explants by electroporation. Curr Protoc Neurosci Chapter 4:Unit 4.34.1–10

    Google Scholar 

  11. Parker M, Brugeaud A, Edge AS (2010) Primary culture and plasmid electroporation of the murine organ of Corti. J Vis Exp 36:1685

    PubMed  Google Scholar 

  12. Masuda M, Pak K, Chavez E, Ryan AF (2012) TFE2 and GATA3 enhance induction of POU4F3 and myosin VIIa positive cells in nonsensory cochlear epithelium by ATOH1. Dev Biol 372:68–80

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Haque KD, Pandey AK, Kelley MW, Puligilla C (2015) Culture of embryonic mouse cochlear explants and gene transfer by electroporation. J Vis Exp 95:52260

    PubMed  Google Scholar 

  14. Jones JM, Montcouquiol M, Dabdoub A, Woods C, Kelley MW (2006) Inhibitors of differentiation and DNA binding (Ids) regulate Math1 and hair cell formation during the development of the organ of Corti. J Neurosci 26:550–558

    Article  CAS  PubMed  Google Scholar 

  15. Schneider ME, Belyantseva IA, Azevedo RB, Kachar B (2002) Rapid renewal of auditory hair bundles. Nature 418:837–838

    Article  CAS  PubMed  Google Scholar 

  16. Belyantseva IA, Boger ET, Friedman TB (2003) Myosin XVa localizes to the tips of inner ear sensory cell stereocilia and is essential for staircase formation of the hair bundle. Proc Natl Acad Sci U S A 100:13958–13963

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Rzadzinska AK, Schneider ME, Davies C, Riordan GP, Kachar B (2004) An actin molecular treadmill and myosins maintain stereocilia functional architecture and self-renewal. J Cell Biol 164:887–897

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Belyantseva IA, Boger ET, Naz S, Frolenkov GI, Sellers JR, Ahmed ZM et al (2005) Myosin-XVa is required for tip localization of whirlin and differential elongation of hair-cell stereocilia. Nat Cell Biol 7:148–156

    Article  CAS  PubMed  Google Scholar 

  19. Boger ET, Frolenkov GI, Friedman TB, Belyantseva IA (2008) Myosin XVa. In: Coluccio LM (ed) Myosins: a superfamily of molecular motors. Springer, The Netherlands, pp 441–467

    Google Scholar 

  20. Geng R, Melki S, Chen DH et al (2012) The mechanosensory structure of the hair cell requires clarin-1, a protein encoded by Usher syndrome III causative gene. J Neurosci 32:9485–9498

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Grati M, Kachar B (2011) Myosin VIIa and sans localization at stereocilia upper tip-link density implicates these Usher syndrome proteins in mechanotransduction. Proc Natl Acad Sci U S A 108:11476–11481

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Zhao H, Avenarius MR, Gillespie PG (2012) Improved biolistic transfection of hair cells. PLoS One 7:e46765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Morín M, Bryan KE, Mayo-Merino F et al (2009) In vivo and in vitro effects of two novel gamma-actin (ACTG1) mutations that cause DFNA20/26 hearing impairment. Hum Mol Genet 18:3075–3089

    Article  PubMed  PubMed Central  Google Scholar 

  24. Kawashima Y, Géléoc GS, Kurima K et al (2011) Mechanotransduction in mouse inner ear hair cells requires transmembrane channel-like genes. J Clin Invest 121:4796–4809

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Diaz-Horta O, Subasioglu-Uzak A, Grati M et al (2014) FAM65B is a membrane-associated protein of hair cell stereocilia required for hearing. Proc Natl Acad Sci U S A 111: 9864–9868

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Felgner PL, Gadek TR, Holm M et al (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A 84:7413–7417

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Weaver JC (1995) Electroporation theory. Concepts and mechanisms. Methods Mol Biol 47:1–26

    CAS  PubMed  Google Scholar 

  28. Favard C, Dean DS, Rols MP (2007) Electrotransfer as a non viral method of gene delivery. Curr Gene Ther 7:67–77

    Article  CAS  PubMed  Google Scholar 

  29. Saito T, Nakatsuji N (2001) Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev Biol 240:237–246

    Article  CAS  PubMed  Google Scholar 

  30. Wang W, Liu X, Gelinas D, Ciruna B, Sun Y (2007) A fully automated robotic system for microinjection of zebrafish embryos. PLoS One 2:e862

    Article  PubMed  PubMed Central  Google Scholar 

  31. Gonzalez-Estevez C, Momose T, Gehring WJ, Salo E (2003) Transgenic planarian lines obtained by electroporation using transposon-derived vectors and an eye-specific GFP marker. Proc Natl Acad Sci U S A 100: 14046–14051

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kunieda T, Kubo T (2004) In vivo gene transfer into the adult honeybee brain by using electroporation. Biochem Biophys Res Commun 318:25–31

    Article  CAS  PubMed  Google Scholar 

  33. Ando T, Fujiwara H (2013) Electroporation-mediated somatic transgenesis for rapid functional analysis in insects. Development 140: 454–458

    Article  CAS  PubMed  Google Scholar 

  34. Matsumoto CS, Shidara H, Matsuda K et al (2013) Targeted gene delivery in the cricket brain, using in vivo electroporation. J Insect Physiol 59:1235–1241

    Article  CAS  PubMed  Google Scholar 

  35. Klein TM, Wolf ED, Wu R, Sanford JC (1987) High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327: 70–73

    Article  CAS  Google Scholar 

  36. Klein TM, Fromm M, Weissinger A et al (1988) Transfer of foreign genes into intact maize cells with high-velocity microprojectiles. Proc Natl Acad Sci U S A 85:4305–4309

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Zelenin AV, Titomirov AV, Kolesnikov VA (1989) Genetic transformation of mouse cultured cells with the help of high-velocity mechanical DNA injection. FEBS Lett 244:65–67

    Article  CAS  PubMed  Google Scholar 

  38. Johnston SA (1990) Biolistic transformation: microbes to mice. Nature 346:776–777

    Article  CAS  PubMed  Google Scholar 

  39. Yang NS, Burkholder J, Roberts B, Martinell B, McCabe D (1990) In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment. Proc Natl Acad Sci U S A 87:9568–9572

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Williams RS, Johnston SA, Riedy M, DeVit MJ, McElligott SG, Sanford JC (1991) Introduction of foreign genes into tissues of living mice by DNA-coated microprojectiles. Proc Natl Acad Sci U S A 88:2726–2730

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Sanford JC, Smith FD, Russell JA (1993) Optimizing the biolistic process for different biological applications. Methods Enzymol 217:483–509

    Article  CAS  PubMed  Google Scholar 

  42. O’Brien JA, Lummis SC (2002) An improved method of preparing microcarriers for biolistic transfection. Brain Res Protoc 10:12–15

    Article  Google Scholar 

  43. Thomas JL, Bardou J, L’hoste S, Mauchamp B, Chavancy G (2001) A helium burst biolistic device adapted to penetrate fragile insect tissues. J Insect Sci 1:9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kim TW, Lee JH, He L et al (2005) Modification of professional antigen-presenting cells with small interfering RNA in vivo to enhance cancer vaccine potency. Cancer Res 65:309–316

    CAS  PubMed  Google Scholar 

  45. Pascolo S (2006) Vaccination with messenger RNA. Methods Mol Med 127:23–40, Review

    CAS  PubMed  Google Scholar 

  46. Yang CH, Shen SC, Lee JC et al (2004) Seeing the gene therapy: application of gene gun technique to transfect and decolour pigmented rat skin with human agouti signaling protein cDNA. Gene Ther 11:1033–1039

    Article  CAS  PubMed  Google Scholar 

  47. Shefi O, Simonnet C, Baker MW, Glass JR, Macagno ER, Groisman A (2006) Microtargeted gene silencing and ectopic expression in live embryos using biolistic delivery with a pneumatic capillary gun. J Neurosci 26:6119–6123

    Article  CAS  PubMed  Google Scholar 

  48. O’Brien JA, Lummis SC (2006) Biolistic transfection of neuronal cultures using a hand-held gene gun. Nat Protoc 1:977–981

    Article  PubMed  PubMed Central  Google Scholar 

  49. Drummond MC, Barzik M, Bird JE et al (2015) Live-cell imaging of actin dynamics reveals mechanisms of stereocilia length regulation in the inner ear. Nat Commun 6:6873

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Zhang DS, Piazza V, Perrin BJ et al (2012) Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia. Nature 481:520–524

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Belyantseva IA (2013) CLIC5 interacts with taperin at the base of stereocilia. In: Abstracts of the 9th molecular biology of hearing and deafness, Stanford University, Palo Alto, CA, Accessed 22–25 June

    Google Scholar 

  52. Furness DN, Mahendrasingam S, Ohashi M, Fettiplace R, Hackney CM (2008) The dimensions and composition of stereociliary rootlets in mammalian cochlear hair cells: comparison between high- and low-frequency cells and evidence for a connection to the lateral membrane. J Neurosci 28(25):6342–6353

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kitajiri S, Sakamoto T, Belyantseva IA et al (2010) Actin-bundling protein TRIOBP forms resilient rootlets of hair cell stereocilia essential for hearing. Cell 141:786–798

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Di Pasquale G, Rzadzinska A, Schneider ME, Bossis I, Chiorini JA, Kachar B (2005) A novel bovine virus efficiently transduces inner ear neuroepithelial cells. Mol Ther 11:849–855

    Article  PubMed  Google Scholar 

  55. Probst FJ, Fridell RA, Raphael Y et al (1998) Correction of deafness in shaker-2 mice by an unconventional myosin in a BAC transgene. Science 280:1444–1447

    Article  CAS  PubMed  Google Scholar 

  56. Holme RH, Kiernan BW, Brown SD, Steel KP (2002) Elongation of hair cell stereocilia is defective in the mouse mutant whirler. J Comp Neurol 450:94–102

    Article  PubMed  Google Scholar 

  57. Mburu P, Mustapha M, Varela A et al (2003) Defects in whirlin, a PDZ domain molecule involved in stereocilia elongation, cause deafness in the whirler mouse and families with DFNB31. Nature Genet 34:421–428

    Article  CAS  PubMed  Google Scholar 

  58. Delprat B, Michel V, Goodyear R et al (2005) Myosin XVa and whirlin, two deafness gene products required for hair bundle growth, are located at the stereocilia tips and interact directly. Hum Mol Genet 14:401–410

    Article  CAS  PubMed  Google Scholar 

  59. Gagnon LH, Longo-Guess CM, Berryman M et al (2006) The chloride intracellular channel protein CLIC5 is expressed at high levels in hair cell stereocilia and is essential for normal inner ear function. J Neurosci 26:10188–10198

    Article  CAS  PubMed  Google Scholar 

  60. Salles FT, Andrade LR, Tanda S et al (2014) CLIC5 stabilizes membrane-actin filament linkages at the base of hair cell stereocilia in a molecular complex with radixin, taperin, and myosin VI. Cytoskeleton 71:61–78

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. O’Brien JA, Lummis SC (2011) Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles. BMC Biotechnol 11:66

    Article  PubMed  PubMed Central  Google Scholar 

  62. Arsenault J, O’Brien JA (2013) Optimized heterologous transfection of viable adult organotypic brain slices using an enhanced gene gun. BMC Res Notes 6:544

    Article  PubMed  PubMed Central  Google Scholar 

  63. Arsenault J, Nagy A, Henderson JT, O’Brien JA (2014) Regioselective biolistic targeting in organotypic brain slices using a modified gene gun. J Vis Exp 92:e52148

    PubMed  Google Scholar 

  64. Russell JA, Roy MK, Sanford JC (1992) Physical trauma and tungsten toxicity reduce the efficiency of biolistic transformation. Plant Physiol 98:1050–1056

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Belyantseva IA (2009) Helios Gene Gun-mediated transfection of the inner ear sensory epithelium. In: Sokolowski B (ed) Auditory and vestibular research. Methods and protocols. Methods Mol Biol 493, Humana Press, Springer, Heidelberg, pp 103–124

    Google Scholar 

Download references

Acknowledgement

I thank Thomas Friedman for support and encouragement as well as for critical reading of this chapter, Jonathan Gale (University of London, UK) for teaching me the rat organ of Corti explant technique, Erich Boger for preparing myosin 15a and whirlin cDNA expression constructs, Atteeq Rehman for the Clic5-GFP construct; Gregory Frolenkov and Doris Wu for critical reading of the manuscript and helpful discussions. This work was supported by funds from the NIDCD Intramural Program (1ZIADC000039 and 1ZIADC000048) to Thomas B. Friedman.

Author information

Authors and Affiliations

  1. Laboratory of Molecular Genetics, Section on Human Genetics, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, MD, 20892-3729, USA

    Inna A. Belyantseva

Authors
  1. Inna A. Belyantseva
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Inna A. Belyantseva .

Editor information

Editors and Affiliations

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

    Bernd Sokolowski

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Belyantseva, I.A. (2016). Helios® Gene Gun-Mediated Transfection of the Inner Ear Sensory Epithelium: Recent Updates. 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_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3615-1_1

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3613-7

  • Online ISBN: 978-1-4939-3615-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation