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Laboratory Techniques

Overview

We study various aspects of stroke and spinal cord injury with the aim of understanding molecular events and, eventually, helping develop therapies for these neurological injuries.

If you would like further information about any of the techniques that my laboratory has experience using, or if you would like conduct research in collaboration with my laboratory, please don’t hesitate to get in contact.

Background

Various processes within neurons control their ability to extend an axon after injury. These processes occur at the level of gene expression (transcription of DNAs into RNAs) and translation (conversion of RNAs into proteins). We analyse gene expression and protein distribution using complementary methods: 

Analysis of gene expression

  • Nucleic acid extraction and purification
  • RNA amplification (T7 based)
  • Biotin labelling of samples for microarray hybridisation
  • Microarray analysis (dChip, Onto Tools)
  • Real time reverse transcription (RT) polymerase chain reaction (PCR)
  • Northern blot

Analysis of protein expression

  • Immunolabelling
  • Western blotting
  • Enzyme linked immunosorbent assay (ELISA)

We have used these approaches to identify numerous genes or proteins whose abundances correlate with axon growth. Together with collaborators in the USA , we are using in vitro approaches to determine which of these molecules play key causal roles in controlling axon growth.

These methods have identified potential new targets for promoting growth of injured axons in the brain and spinal cord. Work by other groups has also identified a range of other potential targets for nervous system repair.

 

In order to control the expression level and locus of potential new growth-promoting molecules, we are developing methods for targeted manipulation of gene expression. This involves genetically modifying various viral vectors for gene overexpression and gene knockdown.

In vitro methodologies

  • Derivation, purification and maintenance of cell lines and primary cells including postnatal cerebellar granule neurons and spinal cord neurons.
  • Manipulation of gene expression
  • Cloning
  • Plasmid-based RNA interference (pSUPER)
  • Plasmid-based gene overexpression (pCMV-SPORT)
  • Viral vectors for gene overexpression (Lentivirus)
  • Viral vectors for RNA interference (ShLenti)

Our ultimate goal is to identify methods for promoting recovery of function in humans. An intermediate step often involves testing of potential therapies in animal models of disease or injury.

In vivo methodologies including behavioural testing

  • Surgery
  • Tracing
  • Behavioural testing
  • Histology
  • Tissue preparation, cryostat and freezing microtome sectioning
  • Immunohistochemistry
  • Microscopy
  • Analysis

 

Quick Links

Nature Protocols manuscript and links

Publications as PDFs

Media coverage as PDFs