Lentiviral transfection of ependymal primary cultures facilitates the characterisation of kinocilia-specific promoters.
Ependymal primary cultures (EPCs) are an established model for studying ependymal cell biochemistry and the biology of kinocilia-bearing cells. However, the difficulty in causing them to express transgenes at high efficiency has been an important drawback of the system. Indeed plasmid-based transfection attempts remain at an efficiency below 1% and fail to elicit reporter gene expression, namely green fluorescent protein (GFP) synthesis, in any of the kinocilia-bearing cells of the cultures. Human immunodeficiency 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 specifically 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 specifically transcribed in kinocilia-bearing cells, wdr16. The results have identified 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.
Kowtharapu
Bhavani S
BS
Interfaculty Institute for Biochemistry, University of Tuebingen, Hoppe-Seyler-Str. 4, 72076 Tuebingen, Germany.
Vincent
Franklin C
FC
Bubis
Andreas
A
Verleysdonk
Stephan
S
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D002923:Cilia / Q000502:physiology*; D019295:Computational Biology; D004805:Ependyma / Q000166:cytology*; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D016086:Lentivirus / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000032:analysis / Q000378:metabolism
D014764:Viral Proteins; C079287:scaffold protein, Herpes simplex virus-1; D008156:Luciferases