Proceedings of The Physiological Society
University of York (2002) J Physiol 539P, S009
Subtracted cDNA libraries from regenerating leech ganglia identify transcripts upregulated at different times post-axotomy
R. Emes*, W.-Z. Wang and S.E. Blackshaw
*MRC Functional Genetics Unit and Department of Human Anatomy & Genetics, University of Oxford, Oxford OX1 3QX, UK
In previous experiments, we have constructed cDNA libraries from single types of nerve cells microdissected from Hirudo medicinalis, and have identified genes expressed at 7 days post-axotomy, a relatively late stage of repair when neurons are extending new processes and making synapses (Korneev et al. 1998). Changes in gene expression as a result of axotomy clearly occur at earlier times after damage, and we have now extended this approach to study gene expression at earlier stages of repair. In these experiments we have used whole ganglia rather than microdissected single neurons for the construction of libraries. Each segmental ganglion consists of around 400 neurons together with macro- and microglia and the cells of the connective tissue sheath of the ganglion. The subtractive libraries will therefore represent the differentially expressed genes of all cells that may contribute to the repair process.
We have constructed cDNA libraries from non-regenerating adult ganglia and from regenerating ganglia at 24, 72 and 120 h post-axotomy. For each regenerating ganglion cDNA library the paired nerve roots of the 9th segmental ganglion from three leeches were cut in leeches anaesthetised in 0.15 % w/v chlorobutanol and then allowed to regenerate before the ganglia were removed for extraction of RNA. Non-regenerating cDNA was derived from the 9th segmental ganglion of three leeches that was immediately removed from leeches after sham operation. After isolation of total RNA, first strand cDNA was produced. An oligo-dG tail was added and second strand cDNA was synthesized. Polymerase chain reaction (PCR) was then used to amplify sufficient cDNA to clone into a Lambda vector. From this material subtracted libraries were made using suppressor PCR technology to isolate genes upregulated at the chosen times after axotomy.
Sequencing of selected clones from the 24 h subtractive library has to date identified the following clones with known homologies. Two clones were cytochrome oxidase subunit II, and 16s ribosomal RNA, both found in the mitochondrial genome. A third clone was identified as the valine-rich protein first isolated as a novel clone in a subtractive library from leech serotonergic Retzius neurons at 7 days of regeneration (Accession no. AAB40925; Korneev et al. 1998). This clone was subsequently shown to be a homologue of the SPTR protein identified in serotonergic neurons in Lymnaea and thought to function as a co-transmitter in the snail CNS (Accession no. AF129397; Koert et al. 2001).
In summary, this approach enables us to identify transcripts differentially expressed in a defined neuronal population at early stages after damage and during repair. The subtracted cDNA can subsequently be used to screen single neuron libraries in order to isolate those genes that are up- or downregulated in defined cell types (Wang et al. 2002).
This work is supported by the MRC and a HFSP grant to S.E.B.
Where applicable, experiments conform with Society ethical requirements