The calretinin (CR) knockout project, initiated by Dr. Strauss, has been taken over by Drs. Kawagoe and Winsky under the guidance of Dr. Porter. Southern blot analyses are underway to identify 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). Promoter analyses of the CR gene using a 1.6 kbp region upstream of exon 1 are continuing. Sequence analyses of this putative promoter region reveled a single SP1 site, a single CAAT box and a TATA box. Examination of promoter gene expression (using luciferase and beta-galactocidase) revealed putative silencer and enhancer sequences within the promoter region. Preliminary gel mobility shift assays (GMSA) by Dr. Strauss revealed region-specific changes in the mobility of pieces of promoter sequence. Additional GMSAs using smaller promoter sequences by Dr. Winsky and Mr. Wolpoe in collaboration with Dr. Iadorola revealed several shifts that were specific for nuclear extracts of mouse brain (+ CR expressing cells) or liver (- CR expressing cells). CR transgenic mice were developed using a construct containing CR cDNA under control of three tandem repeats of the T-alpha-1 tubulin promoter for examining the effects of CR over-expression in CNS neurons during development in C57BL/6-C3H mice. Six transgenic offspring were identified by PCR analysis. Behavioral testing of F1 generation offspring of transgenic mice mated with normal C57BL/6-C3H mice revealed an increase in activity in dark period open field tests. Animals are being evaluated to examine the duration of CR over-expression and the possibility that hyperactivity in transgenic mice may be due to CR-induced disruption of the dopamine system during development.
