The CR knockout mouse project has been advanced using an 18 kbp clone containing calretinin (CR) exon 1, 7 kbp 5' sequences and 11 kbp 3' intronic sequences. Approximately 3-4 kb have been sequenced. A targeting plasmid construct has been completed. Homologous recombination would replace 925 bp containing CR exon 1 with pGK-neo (about 1.4 kbp). With Dr. F.D. Porter (Dr. Westphal's lab), the construct was electroporated into embryonic stem cells that were double selected. Colonies were harvested, expanded, and half the cells were frozen while the other half were used to make DNA for analysis. Southern blot results were inconclusive; there were no signals from the CR probes or probes provided by Dr. Porter. Therefore, PCR analyses are in progress. Promoter analysis of the CR gene utilizes a 1.6 kbp region just upstream of exon 1. A combination of elements identify this as the putative promoter region. There exist a single SP1 site (at -139 bp from the translation start site), a single CAAT box at position -76, and a TATA box at position -57. Analyses of both functional activity (reporter gene expression) and protein binding (gel mobility shift assays, GMSA) of this region are being carried out. GMSA data confirm the SP1 site at position -139. Portions of the 1.6 kbp promoter show specific protein binding (or a lack thereof) in mouse nuclear extracts. To characterize the in vivo activity of these sequences, we subcloned the 1.6 kbp region and portions of it into reporter vectors upstream of either the luciferase or beta- galactosidase coding region. Preliminary results show that the 1.6 kbp region as a whole does not promote luciferase expression. To detect upregulation of this gene, transfection of these vectors into CR expressing cultured embryonic mouse brain cells is in progress. Other findings PC-12 cells have been transfected with a plasmid containing the CR coding sequence under the control of a CMV promoter. We are investigating whether ectopic CR expression affects neuronal survival, differentiation, or apoptosis. The cells are being observed before and after NGF-induced differentiation. Finally, we discovered that CR, LH receptor, and testosterone are expressed in concert during male rats' first 6 weeks. FSH receptor and beta-actin are expressed with an altogether different parallel time course. This may indicate that CR is associated with Leydig cell ontogeny and possibly regulation of testosterone production in the testes.