Transfection of Mouse Cochlear Explants by Electroporation

Driver, Elizabeth; Kelley, Matthew W.

doi:10.1002/0471142301.ns0434s51

Cited by 21 publications

(19 citation statements)

References 20 publications

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“…With the electroporation method used, the majority of transfected cells observed in P1.5 cochlear epithelial samples were located in the GER, medial to the sensory cell region, as has been reported previously (Zheng and Gao, 2000; Jones et al, 2006; Driver and Kelley, 2010; Zhao et al, 2011). Cells lateral to the HCs or HCs themselves were occasionally transfected, but this was rare.…”

Section: Resultssupporting

confidence: 58%

TFE2 and GATA3 enhance induction of POU4F3 and myosin VIIa positive cells in nonsensory cochlear epithelium by ATOH1

Masuda¹,

Pak

Chávez³

et al. 2012

Developmental Biology

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Transcription factors (TFs) can regulate different sets of genes to determine specific cell types by means of combinatorial codes. We previously identified closely-spaced TF binding motifs located 8.2-8.5 kb 5′ to the ATG of the murine Pou4f3 gene, a gene required for late hair cell (HC) differentiation and survival. These motifs, 100% conserved among four mammalian species, include a cluster of E-boxes preferred by TCF3/ATOH1 heterodimers as well as motifs for GATA factors and SP1. We hypothesized that these factors might interact to regulate the Pou4f3 gene and possibly induce a HC phenotype in non-sensory cells of the cochlea. Cochlear sensory epithelium explants were prepared from postnatal day 1.5 transgenic mice in which expression of GFP is driven by 8.5 kb of Pou4f3 5′ genomic DNA (Pou4f3/GFP). Electroporation was used to transfect cells of the greater epithelial ridge with multiple plasmids encoding human ATOH1 (hATOH1), hTCF3 (also known as E2A or TEF2), hGATA3, and hSP1. hATOH1 or hTCF3 alone induced Pou4f3/GFP cells but hGATA3 and hSP1 did not. hATOH1 but not hTCF3 induced conversion of greater epithelial ridge cells into Pou4f3/GFP and myosin VIIa double-positive cells. Transfection of hATOH1 in combination with hTCF3 or hGATA3 induced 2-3X more Pou4f3/GFP cells, and similarly enhanced Pou4f3/GFP and myosin VIIa double-positive cells, when compared to hATOH1 alone. Triple or quadruple TF combinations were generally not more effective than double TF combinations except in the middle turn, where co-transfection of hATOH1, hE2A, and hGATA3 was more effective than hATOH1 plus either hTCF3 or hGATA3. The results demonstrate that TFs can cooperate in regulation of the Pou4f3 gene and in the induction of at least one other element of a HC phenotype. Our data further indicate that combinations of TFs can be more effective than individual TFs in the inner ear.

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Section: Resultssupporting

confidence: 58%

TFE2 and GATA3 enhance induction of POU4F3 and myosin VIIa positive cells in nonsensory cochlear epithelium by ATOH1

Masuda¹,

Pak

Chávez³

et al. 2012

Developmental Biology

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“…P3 C57B6/129SvEv mouse cochleae were dissected and maintained in culture with the aid of Matrigel as previously described ( Driver and Kelley, 2010 ). After cells had been in culture for 1 d, growth medium DMEM (12430-054; GIBCO Life Technologies, with 1% FBS [16000-044; GIBCO Life Technologies] and 50 µg/ml ampicillin) with or without the test compound was added for preincubation for 1 h at 37°C in 5% CO 2 , and this was followed by incubation with 50 µM cisplatin (479306; Sigma-Aldrich) with or without the test compound in growth medium for 24 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

CDK2 inhibitors as candidate therapeutics for cisplatin- and noise-induced hearing loss

Teitz

Fang

Goktug

et al. 2018

Journal of Experimental Medicine

108

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“…Embryos were collected from timed pregnant females and cochlear explant cultures established between embryonic day 13 (E13) and post-natal day 0 (P0) as previously described (Driver and Kelley, 2010; Montcouquiol and Kelley, 2003; Yamamoto et al, 2009). RU486 (Mifepristone, Sigma) was dissolved in DMSO and added to the culture media at the indicated concentration for 1, 4, or 24 hours; the total DMSO concentration was less than or equal to 0.2%.…”

Section: Methodsmentioning

confidence: 99%

The Atoh1-lineage gives rise to hair cells and supporting cells within the mammalian cochlea

Driver

Sillers

Coate

et al. 2013

Developmental Biology

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123

132

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The Organ of Corti, located within the mammalian cochlea, contains a precise mosaic of hair cells (HC) and supporting cells (SC), the patterning of which is critical for auditory function. Progenitors of HCs and SCs are found in the same post-mitotic region of the cochlear duct during early stages of cochlear development, and both HCs and SCs are absent in mice lacking the transcription factor Atoh1. Based on existing data, Atoh1 is thought to be the earliest determinant of HC fate, and to have a cell-autonomous role in HC differentiation, but the lineage of Atoh1-positive cells within the cochlear duct remains unclear. To address this issue, we used an inducible Atoh1Cre*PR allele to permanently mark Atoh1-expressing cells at different developmental time points. We found that up to 30% of cells from the Atoh1-lineage develop as SCs, and that the number of Atoh1-positive SCs decreases both spatially and temporally in a pattern consistent with ongoing commitment. Modulation of Notch signaling, necessary for formation of the HC-SC mosaic, changes the percentage of cells from the Atoh1-lineage that develop as either HCs or SCs. The HC-SC ratio is also affected by morphogenesis of the cochlea, as inhibiting the outgrowth of the cochlear duct increases the number of Atoh1-lineage cells that develop as SCs. Our results demonstrate that the Atoh1-lineage is established early in cochlear development, but also show that expression of Atoh1 does not absolutely result in commitment to a HC fate.

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Transfection of Mouse Cochlear Explants by Electroporation

Cited by 21 publications

References 20 publications

TFE2 and GATA3 enhance induction of POU4F3 and myosin VIIa positive cells in nonsensory cochlear epithelium by ATOH1

TFE2 and GATA3 enhance induction of POU4F3 and myosin VIIa positive cells in nonsensory cochlear epithelium by ATOH1

CDK2 inhibitors as candidate therapeutics for cisplatin- and noise-induced hearing loss

The Atoh1-lineage gives rise to hair cells and supporting cells within the mammalian cochlea

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