CDNA LIBRARIES FROM A FEW NEURAL CELLS

One of the most powerful approaches to the molecular analysis of diffe rential gene expression is to construct cDNA libraries corresponding t o different tissues or developmental stages, and then to enrich for ge nes expressed in a particular tissue or at a particular time by subtra ctive hybridisation. Our aim is to reduce the complexity of neuronal c DNA libraries by generating libraries from the mRNA of a single cell. The system chosen is the Retzius cell of the leech, a large neurone wh ich can be unambiguously dissected out. A cDNA library was generated f rom one leech ganglion (containing about 400 neurons) by anchor 1-olig o 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 r egions. XBaI and Eco RI sites were included in the respective anchor p rimers, between the dT or dC run and the PCR primer sequence, allowing high-efficiency directional cloning. Eight clones picked and sequence d 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% o f the clones hybridised to repetitive DNA, and 20/30,000 clones gave s ignals with the Drosophila actin gene. This approach has now been exte nded to a few pooled Retzius cells.