A novel approach to identify proteins associated with the inhibition of neurite growth.
In the present study, a novel combination of techniques was used to identify the genes that may be involved in the lack of axonal regeneration in the mammalian adult central nervous system (CNS). The key features of this approach are: (1) a functional assay that can be affected by antibody perturbation; (2) increased specificity of the polyclonal antiserum by adsorption; (3) the expression cloning of the genes from a lambdagt11 library; (4) amplification of the insert cDNA by PCR; and (5) the direct cycle sequencing of PCR products. In this culture assay system, neurons were plated directly on sections of the rat CNS. This assay system could be used to demonstrate the lack of neuronal attachment to or neurite extension over myelinated regions of the CNS (white matter). This prohibitive nature of the CNS sections could be masked by a rabbit polyclonal antiserum directed against rat CNS white matter. This data indicates that the anti-white matter antiserum recognizes and neutralizes inhibitory molecules on the surface of the sections. Making the assumption that the prohibitive antigen is associated with the cell membrane, the antiserum was adsorbed against a soluble protein fraction of the adult rat brain. This adsorption significantly increased the specificity of the antiserum as demonstrated by immunoblot methods. The adsorbed antiserum was then used to screen the cDNA library of the adult rat brain. The present report describes this novel combination of techniques allowing one to go from a functional tissue culture assay system to defining the molecular basis for the cellular interactions.
Geisert
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EE
Department of Anatomy and Neurobiology, University of Tennessee, Memphis 38163, USA. egeisert@nb.utmem.edu
Seo
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H
Sullivan
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CD
Yang
L J
LJ
Grefe
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A
D000327:Adsorption; D000818:Animals; D003001:Cloning, Molecular; D003337:Corpus Callosum / Q000502:physiology; D005260:Female; D015870:Gene Expression / Q000276:immunology; D006131:Growth Inhibitors / Q000502:physiology*; D006624:Hippocampus / Q000502:physiology; D007106:Immune Sera / Q000502:physiology; D008297:Male; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D009433:Neural Inhibition / Q000187:drug effects / Q000235:genetics; D016501:Neurites / Q000187:drug effects / Q000502:physiology*; D009900:Optic Nerve / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley
D006131:Growth Inhibitors; D007106:Immune Sera; D009419:Nerve Tissue Proteins