cDNA libraries from a few neural cells

Abstract

One of the most powerful approaches to the molecular analysis of differential gene expression is to construct cDNA libraries corresponding to different tissues or developmental stages, and then to enrich for genes expressed in a particular tissue or at a particular time by subtractive hybridisation. Our aim is to reduce the complexity of neuronal cDNA libraries by generating libraries from the mRNA of a single cell. The system chosen is the Retzius cell of the leech, a large neurone which can be unambiguously dissected out. A cDNA library was generated from one leech ganglion (containing about 400 neurons) by anchor 1-oligo dT priming, the addition of dG tails, second strand synthesis primed by an anchor 2-oligo dC primer, followed by PCR from the two anchor regions. XBaI and EcoRI sites were included in the respective anchor primers, between the dT or dC run and the PCR primer sequence, allowing high-efficiency directional cloning. Eight clones picked and sequenced at random gave five with some homology to a known protein and three novel genes. The average insert size in the library was 600 bp, 0.2% of the clones hybridised to repetitive DNA, and 20/30,000 clones gave signals with the Drosophila actin gene. This approach has now been extended to a few pooled Retzius cells.