Differential display cloning of genes induced in regenerating neurons.
Mammalian adult motor and sensory neurons are thought to reexpress a complement of genes that are originally expressed during development when they regenerate following injury. Therefore, one strategy for identifying key genes involved in development of the peripheral nervous system is to identify those genes reexpressed in the regenerating system. To test this hypothesis, we used the single-base anchor method of mRNA differential display to study changes in gene expression in regenerating adult mammalian sensory neurons. From an initial sample of 36 different primer combinations [3 oligo(dT)M primers x 12 arbitrary 13-mers], 6 candidate upregulated and 6 candidate downregulated genes were identified. Candidate genes were screened by the reverse Northern blot method to eliminate obvious false positives and the three remaining candidates cloned and sequenced. In addition to comparing isolated sequences with the public databases, sequences were also compared with assembled clusters of expressed sequence tag sequences, enabling extension of the sequence data by more than a kilobase from the isolated 3' cDNA fragments. Ultimate confirmation of differential expression was carried out by in situ hybridization using 45-base oligonucleotides complementary to the predicted 5'-3' orientation of the corresponding mRNAs of all three cDNAs. Two, LA12.2 and LC12, were definitively confirmed as induced in regenerating neurons. The sequence of LC12 is identical to that of the secreted protein Reg-2 and a detailed study of the functions of this secreted protein in neural development and regeneration has been published (F. J. Livesey, J. A. O'Brien, M. Li, A. G. Smith, L. J. Murphy, and S. P. Hunt, 1997, Nature 390, 614-618). The LA12.2 gene is currently being characterized, the available sequence of this cDNA being novel.
Livesey
F J
FJ
Zoology Department, Trinity College, Dublin, Ireland. livesey@rascal.med.harvard.edu
Hunt
S P
SP
D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005727:Ganglia, Spinal / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D017403:In Situ Hybridization; D009416:Nerve Regeneration / Q000235:genetics*; D009475:Neurons, Afferent / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012584:Sciatic Nerve / Q000293:injuries; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger