Cloning and identification of differentially expressed transcripts in primary culture of GABAergic neurons.

A RNA based arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify differentially expressed transcripts in primary cultures of cerebral cortical neurons prepared from E16 mouse cerebral cortex. The majority of neurons found in this culture preparation are known to be GABAergic. Different primer combinations were used, and the PCR products were separated on PAGE. Visualization by silver staining revealed a high resolution RNA fingerprint pattern with a total of about 200 transcripts. Six differentially expressed cDNA fragments were recovered, cloned and sequenced. The results of a NCBI database search showed that 6 clones were highly homologous to known genes and expressed sequence tags (ESTs), and that they were either up-regulated or down-regulated during development. Among these clones, Clone 3.1.7 shared 99% sequence homology to mouse Reelin, a neuronal migration and positioning related protein. Clone 4.6.2 shared 91% homology to Rat prepro bone morphogenetic protein-3 mRNA. Clone 6.10.2 had 90% homology to a novel orphan gene of calcium-independent alpha-latrotoxin receptor, which stimulates presynaptic neurotransmitter release. Northern blot analysis confirmed the up-regulated expression profile of Clone 6.10.2 in neuron from Day 2 to 7 during stages of differentiation and development.