Preparation of nuclear extracts from myelinating Schwann cells.
Myelination of peripheral nerve fibres is performed by Schwann cells and is associated with the coordinate upregulation of lipid synthesis and multiple genes encoding myelin-specific proteins. Both the decision to enter into a myelinating phenotype and subsequently, the quantity of myelin that each Schwann cell elaborates appear to be controlled by axonal signals. Understanding of the relevant signaling pathways and the downstream transcription factors and cis elements that confer myelin gene expression is notably limited. In large part, this has resulted directly from a lack of methods for obtaining nuclear extracts from myelinating Schwann cells thus precluding the application of numerous powerful molecular techniques. In this report, we describe a method that overcomes this limitation for the myelinating Schwann cells in the sciatic nerves of the mouse. During the evolution of the method, its effectiveness was monitored using an oligonucleotide containing the binding site for KROX-20, a transcription factor known to be present in myelinating Schwann cells. Following technical development, the optimized protocol has proven to be entirely reliable and thus novel experimental strategies now can be applied to the investigation of the molecular mechanisms controlling gene expression in peripheral nerves.
Forghani
R
R
Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada.
Nesbitt
J
J
Snipes
J
J
Shooter
E M
EM
Peterson
A
A
D000818:Animals; D000831:Animals, Newborn; D005786:Gene Expression Regulation; D051379:Mice; D009186:Myelin Sheath / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D009687:Nuclear Proteins / Q000032:analysis*; D012583:Schwann Cells / Q000737:chemistry*; D012584:Sciatic Nerve / Q000737:chemistry; D014157:Transcription Factors / Q000032:analysis* / Q000235:genetics
D009687:Nuclear Proteins; D014157:Transcription Factors