MOLECULAR BIOLOGICAL STUDIES OF CALRETININ

作为进一步尝试确定钙视黄素(CR)的功能，我们 已经开展了一项合作努力来准备CR基因敲除小鼠。一个 含有外显子1和5‘侧翼区的CR基因组克隆已被 从C129小鼠文库中分离、限制性内切酶分析和Southern分析 已经完成，5‘区域目前正在进行 序列分析。CR载体(S)的设计与构建 我们实验室正在进行基因敲除小鼠的研究。胚胎的筛选 干细胞、嵌合体和F2后代将由Dr。 施特劳斯。胚胎干细胞转化、胚泡注射、植入和 将与丹尼·波特博士一起进行老鼠饲养 在海纳·威斯特法尔博士的实验室里。 使用固体组织裂解核糖核酸酶保护分析(RPA)，我们 在几个脑区发育的CRmRNA已经被测量在 外科手术、药物治疗和饮食治疗。对大鼠进行治疗 皮质酮(0.1、1、10 mg/d，7d)。下降了93% 在下丘脑室旁核观察到，减少了40% 在医学上被观察到。杏仁核和团粒扁桃核最高 剂量。β-肌动蛋白mRNA(内标)不受 皮质酮。摘除肾上腺不改变任何一组枯萎的mRNA。 检测了一些区域。在睾丸中，观察到以下剂量反应 皮质酮治疗。CR蛋白分别下降了81%、68%和39% 剂量分别为10 mg/d、1 mg/d和0.1 mg/d。 在一项全面的调查中，在睾丸中发现了CR 使用微穿孔RPA对大鼠固体组织进行观察。组织化学技术 证实其存在于间质细胞中。间质细胞 产生睾酮，这种激素也受大剂量皮质酮的影响 (下降%)。睾丸间质细胞对睾酮的发育 成熟期发生在胚胎第16天和出生后。 6周龄幼鼠的睾酮和CR的研究(出生后第3天-- 42)表现出平行的浓度分布。从高起点开始 第3天CR蛋白和mRNA以及睾酮的浓度 浓度在7-14天之间降至最低点，再次上升至 中间浓度随后在大约42天内趋于平稳。 日粮缺钙对28%仔猪CR基因表达的影响 黑质致密带腹侧被盖减少。 区域。酪氨酸羟基酶和β-肌动蛋白mRNA均未受影响。 在测试的其他5个大脑区域也没有观察到任何变化。这 地区性降低可能与寻钙行为有关 这些动物所展示的。

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.