MOLECULAR BIOLOGICAL STUDIES OF CALRETININ

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As a further attempt to determine the function of calretinin (CR), we have undertaken a collaborative effort to prepare CR knockout mice. A CR genomic clone containing exon 1 and 5' flanking regions has been isolated from a C129 mouse library, restriction and Southern analyses have been accomplished, and the 5' regions are currently undergoing sequence analyses. The design and construction of vector(s) for CR knockout mice are underway in our laboratory. Screening of Embryonic stem (ES) cells, chimerae, and F2 progeny will be carried out by Dr. Strauss. ES cell transformations, blastocyst injection.implantation, and mouse breeding will be carried out in conjunction with Dr. Denny Porter in Dr. Heiner Westphal's laboratories. Using a solid tissue lysate RNase protection assay (RPA) that we developed the CR mRNA in several brain areas has been measured after surgical, pharmacological and dietary manipulations. Rats were treated with corticosterone (0.1, 1, 10 mg per day, 7 d). A 93% decrease was observed in the hypothalamic paraventricular nucleus, and 40% decreases were observed in the med. amygdaloid n. and n. reuniens at the highest dose. Beta-actin mRNA (internal standard) was unaffected by corticosterone. Adrenalectomy did not change wither mRNA in any of the areas assayed. In the testes, a dose response was observed following corticosterone treatment. CR protein was decreased by 81%, 68%, and 39% of vehicle treated controls at doses of 10, 1, and 0.1 mg/d respectively. The discovery of CR in the testes was made during a comprehensive survey of rat solid tissues using the micropunch RPA. Histochemical techniques confirm its presence in the interstitial Leydig cells. Leydig cells produce testosterone which was also affected by high dose corticosterone (64% decrease). The development of Leydig cells to testosterone producing maturity takes place between embryonic day 16 and postnatal week 6. A study of testosterone and CR in young rats (postnatal days 3-- 42) demonstrated parallel concentration profiles. Starting at high concentrations on day 3, both CR protein and mRNA and testosterone concentrations bottomed out between 7---14 days, ascended again to intermediate concentrations then leveled off by approximately 42 days. In a study of dietary calcium deficiency on CR mRNA expression of 28% decrease was detected in the substantia nigra compacta-ventral tegmental area. Neither tyrosine hydroxylase nor beta-actin mRNA were affected, nor were any changes observed in 5 other brain areas tested. This region-specific decrease may relate to the calcium-seeking behavior exhibited by these animals.