Work continues toward the production of a calretinin (CR) knockout mouse. Preliminary Southern blot analyses identified 5 positive clones indicative of homologous recombination of embryonic stem cells with the targeting plasmid which would replace 925 bp containing CR exon 1 with pGK-neo (1.4 kbp). Studies in progress will confirm positive clones and proceed to injection of pseudo-pregnant females for the generation of chimeras. Promoter analyses of the CR gene are continuing. The sequence of the putative promoter region has been published. Examination of promoter gene expression (using luciferase and galactocidase) revealed putative silencer and enhancer sequences within the promoter region. Neuronal specific expression of luciferase reporter was preserved by the 115 bp proximal to the transcription start site for CR. Similar reporter gene (luciferase) activity was supported by CR promoter fragments in PC12 cells that express CR and in primary brain cell cultures. Present studies are aimed at determining the promoter region responsible for specific expression within calretinin containing cells. Additional studies compare the reporter activity of two strains of PC12 cells, one that expresses CR and one that does not, as a first step in identifying essential transcription factors.