Comprehensive screens were made for genes that change their expression during a brief critical period in development when neonatal mammalian central nervous system (CNS) loses its capacity to regenerate.
In newly born opossums older than 12 days regeneration ceases to occur in the cervical spinal cord. It continues for 5 more days in lumbar regions. The mRNA’s expressed in cords that do and do not regenerate were analyzed by polymerase chain reaction-based subtractive hybridization. The mRNAs extracted from cervical cords of animals aged 9 and 12 days were subtracted reciprocally, old from young and young from old.
Additional subtractions were made between lumbar regions of 12 day-old cords (which can regenerate) and cervical regions (which cannot). Mini libraries of approximately 2000 opossum cDNA clones resulted from each subtraction. Many sequences were novel. Others that were expressed differentially were related to cell growth, proliferation, differentiation, motility, adhesion, cytoskeleton and extracellular matrix.
A major task was to narrow the search and to eliminate genes that were not associated with regeneration. Clones from different subtractions were cross-hybridized. After those common to regenerating and nonregenerating cords were rejected, approximately 284 sequences of interest remained. Our results revealed novel sequences, as well as genes involved in transcription, cell signaling, myelin formation, growth cone motility, liver regeneration, and nucleic acid and protein management as the candidates important for neuroregeneration.
For selected genes of potential interest for regeneration (for example cadherin, catenin, myelin basic protein), their temporal and spatial distributions and levels of expression in the CNS were measured by Northern blots, semiquantitative and real-time RT-PCR, and in situ hybridization.
Our experiments set the stage for testing the efficacy of candidate genes in turning on or off the capacity for spinal cord regeneration. Opossum spinal cords in vitro provide a reliable and rapid assay for axon outgrowth and synapse formation.
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