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 ce rebral cortical neurons prepared from E16 mouse cerebral cortex. The majori ty of neurons found in this culture preparation are known to be GABAergic. Different primer combinations were used, and the PCR products were separate d on PAGE. Visualization by silver staining revealed a high resolution RNA fingerprint pattern with a total of about 200 transcripts. Six differential ly expressed cDNA fragments were recovered, cloned and sequenced. The resul ts 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, Clon e 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 pre pro bone morphogenetic protein-3 mRNA. Clone 6.10.2 had 90% homology to a n ovel orphan gene of calcium-independent alpha-latrotoxin receptor, which st imulates presynaptic neurotransmitter release. Northern blot analysis confi rmed the up-regulated expression profile of Clone 6.10.2 in neuron from Day 2 to 7 during stages of differentiation and development.