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Neurochem Res (2009) 34:13801392 DOI 10.1007/s11064-009-9918-7
ORIGINAL PAPER
Lentiviral Transfection of Ependymal Primary Cultures Facilitates the Characterisation of Kinocilia-specic Promoters
Bhavani S. Kowtharapu Franklin C. Vincent Andreas Bubis Stephan Verleysdonk
Accepted: 17 January 2009 / Published online: 4 February 2009 Springer Science+Business Media, LLC 2009
Abstract Ependymal primary cultures (EPCs) are an established model for studying ependymal cell biochemistry and the biology of kinocilia-bearing cells. However, the difculty in causing them to express transgenes at high efciency has been an important drawback of the system. Indeed plasmid-based transfection attempts remain at an efciency below 1% and fail to elicit reporter gene expression, namely green uorescent protein (GFP) synthesis, in any of the kinocilia-bearing cells of the cultures. Human immunodeciency virus pseudotyped with the vesicular stomatitis virus envelope glycoprotein (HIV/ VSV-G) and encoding GFP under the control of the ubiquitously recognised promoter of elongation factor 1 alpha (EF1alpha) also does not cause transgene expression in the kinocilia-bearing cells of an EPC when applied at multiplicities of infection (MOIs) of up to 40 and destroys the culture when the MOI is increased further. In contrast, HIV/VSV-G encoding GFP under the control of a promoter specically active in kinocilia-bearing cells leads to transgene expression in up to 79% of the kinociliated cells of an EPC when applied at an MOI of 20. This has permitted the initial characterisation of the promoter for the gene specically transcribed in kinocilia-bearing cells, wdr16. The results have identied two regions of 100
nucleotides length each, which are critical for promoter activity and contain putative binding sites for the transcription factors Foxd1, Sox17 and Spz1. It appears that wdr16 is controlled by a bidirectional promoter also responsible for regulating the syntaxin 8 gene.
Keywords wdr16 HIV Pseudotype Gene transfer
Brain Glia Cerebral ventricles WDPUH
Introduction
Kinocilia are phylogenetically ancient [11, 42] motile cell appendages involved in important cellular processes such as locomotion, creation of laminar or circular uid ows over epithelial surfaces [35] and generation as well as maintenance of planar cell polarity [37]. They generally have an internal skeleton of nine microtubule doublets arranged in a circle and connected by dynein arms in which the ATPase responsible for force generation and axonemal movement resides [50]. The type of kinocilia which does not...