Transfection of Mouse Cochlear Explants by Electroporation

National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland, USA.
Current protocols in neuroscience / editorial board, Jacqueline N. Crawley ... [et al.] 04/2010; Chapter 4:Unit 4.34.1-10. DOI: 10.1002/0471142301.ns0434s51
Source: PubMed


The sensory epithelium of the mammalian inner ear, also referred to as the organ of Corti, is a remarkable structure comprised of highly ordered rows of mechanosensory hair cells and non-sensory supporting cells located within the coiled cochlea. This unit describes an in vitro explant culture technique that can be coupled with gene transfer via electroporation to study the effects of altering gene expression during development of the organ of Corti. While the protocol is largely focused on embryonic cochlea, the same basic protocol can be used on cochleae from mice as old as P5.

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    • "epithelial explants with plasmid encoding dsRed driven by a constitutive (CMV) promoter. 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. "
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    ABSTRACT: 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 8.2-8.5kb 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 8.5kb 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.
    Full-text · Article · Sep 2012 · Developmental Biology
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