Using injectoporation to deliver genes to mechanosensory hair cells

Xiong, Wei; Wagner, Thomas F.J.; Yan, Linxuan; Grillet, Nicolas; Müller, Ulrich

doi:10.1038/nprot.2014.168

Cited by 43 publications

(61 citation statements)

References 65 publications

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“…Primary antibodies were: anti-LHFPL5 (35); anti-PCDH15 PB811 (4); anti-Myc (Cell Signaling); and anti-GFP (14). Hair cells in cochlear cultures were transfected with Myc-Tmc2 containing plasmid using injectoporation (14,39). Cochlear segments from P4 mice were cultured in DMEM/F12 medium with 1.5 mg/mL ampicillin, and the plasmid (1 μg/mL in HBSS) was delivered to the epithelium with a glass pipette.…”

Section: Methodsmentioning

confidence: 99%

Subunit determination of the conductance of hair-cell mechanotransducer channels

Beurg

Xiong

Zhao

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Cochlear hair cells convert sound stimuli into electrical signals by gating of mechanically sensitive ion channels in their stereociliary (hair) bundle. The molecular identity of this ion channel is still unclear, but its properties are modulated by accessory proteins. Two such proteins are transmembrane channel-like protein isoform 1 (TMC1) and tetraspan membrane protein of hair cell stereocilia (TMHS, also known as lipoma HMGIC fusion partner-like 5, LHFPL5), both thought to be integral components of the mechanotransduction machinery. Here we show that, in mice harboring an Lhfpl5 null mutation, the unitary conductance of outer hair cell mechanotransducer (MT) channels was reduced relative to wild type, and the tonotopic gradient in conductance, where channels from the cochlear base are nearly twice as conducting as those at the apex, was almost absent. The macroscopic MT current in these mutants was attenuated and the tonotopic gradient in amplitude was also lost, although the current was not completely extinguished. The consequences of Lhfpl5 mutation mirror those due to Tmc1 mutation, suggesting a part of the MT-channel conferring a large and tonotopically variable conductance is similarly disrupted in the absence of Lhfpl5 or Tmc1. Immunolabelling demonstrated TMC1 throughout the stereociliary bundles in wild type but not in Lhfpl5 mutants, implying the channel effect of Lhfpl5 mutations stems from down-regulation of TMC1. Both LHFPL5 and TMC1 were shown to interact with protocadherin-15, a component of the tip link, which applies force to the MT channel. We propose that titration of the TMC1 content of the MT channel sets the gradient in unitary conductance along the cochlea.cochlea | mechanotransducer channels | TMC1 | hair cell | LHFPL5 C ochlear hair cells detect sound stimuli by submicron vibrations of their stereociliary (hair) bundles. The stereocilia are arranged in three to four rows, stepped in height and interconnected by extracellular linkages; the most important for transduction are the tip links (1, 2), composed of cadherin-23 and protocadherin-15 (3, 4). During bundle displacements, they transmit force to activate mechanotransducer (MT) ion channels near the insertion of protocadherin-15 at the lower end of the tip link into the stereociliary tip (5, 6). The molecular identity of the pore-forming subunit of the ion channel is still controversial, but there has been a recent proposal that transmembrane channellike protein isoforms 1 and 2 (TMC1 and TMC2) (7, 8) are possible candidates (9, 10); mutations of these proteins can alter the Ca 2+ selectivity and single-channel conductance of the MT channels, implying that TMC proteins can influence ion conduction through the pore (10-12). However, in Tmc1/Tmc2 double mutants, large mechanically sensitive currents can still be evoked and flow through channels similar to native MT channels (13). Thus, an alternate view is that the TMC1 and TMC2 are accessory but not pore-forming subunits of the channel.Another likely component of the transduction mac...

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Section: Methodsmentioning

confidence: 99%

Subunit determination of the conductance of hair-cell mechanotransducer channels

Beurg

Xiong

Zhao

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

141

194

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“…One new method, "injectoporation" is just at the beginning of its evaluation by different laboratories [ 1 ] and appears to be promising. The technique of intrauterine electroporation is effi cient for hair cell transfection, but is not commonly used, likely because of the special skills required to perform survival surgery on small rodents [ 2 ].…”

Section: Introductionmentioning

confidence: 99%

“…A fully automated robotic system for microinjection was developed and used in zebrafi sh embryos [ 30 ]. Recently, an injectoporation method, combining microinjection of a solution into the sensory epithelium followed quickly by electroporation, was described for the transfection of the mouse organ of Corti [ 1 ]. This method of microinjection followed by electroporation was described previously for transfection of planarians, insect larva, and adult insect brain in vivo [ 31 -34 ].…”

Section: Introductionmentioning

confidence: 99%

“…This method of microinjection followed by electroporation was described previously for transfection of planarians, insect larva, and adult insect brain in vivo [ 31 -34 ]. Xiong and co-authors [ 1 ] adopted this method to transfect auditory hair cells in postnatal mouse organ of Corti explants from postnatal day 0 to postnatal day 4. This method appeared to be effi cient, although effectiveness is decreased with the age of the postnatal mouse organ of Corti explant [ 1 ].…”

Section: Introductionmentioning

confidence: 99%

“…Xiong and co-authors [ 1 ] adopted this method to transfect auditory hair cells in postnatal mouse organ of Corti explants from postnatal day 0 to postnatal day 4. This method appeared to be effi cient, although effectiveness is decreased with the age of the postnatal mouse organ of Corti explant [ 1 ]. The injectoporation procedure requires microinjection skills under an upright microscope and seems well suited for electrophysiology laboratories that study functional responses of transfected cells.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Belyantseva

2016

Methods in Molecular Biology

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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.

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EVL is not essential for cuticular plate and stereocilia development in mouse auditory hair cells

Yan,

Zhang,

et al. 2024

FEBS Letters

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In inner ear hair cells, the stereocilia are inserted into a dense F‐actin‐enriched meshwork named the cuticular plate, which provides support to the stereocilia. Enah/Vasp‐like (EVL) was shown to localize at the cuticular plate, and evl knockdown leads to disrupted cuticular plate and disorganized stereocilia in Xenopus hair cells. In the present work, we show that Evl transcripts are specifically expressed in mouse hair cells, and EVL is localized to the cuticular plate. However, the cuticular plate and stereocilia are unaffected by Evl knockout, and auditory function is largely normal in Evl knockout mice. In conclusion, our present data suggest that EVL is not essential for cuticular plate and stereocilia development in mouse auditory hair cells.

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Using injectoporation to deliver genes to mechanosensory hair cells

Cited by 43 publications

References 65 publications

Subunit determination of the conductance of hair-cell mechanotransducer channels

Subunit determination of the conductance of hair-cell mechanotransducer channels

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

EVL is not essential for cuticular plate and stereocilia development in mouse auditory hair cells

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