MOLECULAR BIOLOGICAL STUDIES OF CALRETININ

CR基因敲除小鼠项目已经使用18KBP的克隆进行了推进 含有钙维A(CR)外显子1、7个kbp 5‘序列和11个kbp 3’序列 内含子序列。大约3-4kb的序列已经被测序。一个 靶向质粒构建已完成。同源重组 将含有CR外显子1的925个碱基替换为pGK-neo(约1.4 kbp)。 对于F.D.波特博士(Westphal博士的实验室)，这个结构是 电穿孔进入经过双重选择的胚胎干细胞。 克隆被收获，扩增，一半的细胞被冷冻，而 另一半被用来制造DNA进行分析。Southern杂交结果 没有定论；没有来自CR探针或探针的信号 由波特博士提供。因此，聚合酶链式反应分析正在进行中。 CR基因的启动子分析利用了恰好在上游的1.6kbp区域 外显子1。一系列元素的组合将其识别为假定的 启动子区域。只有一个SP1位点(在-139BP处 翻译起始点)、位置-76处的单个CAAT框和TATA 盒子在-57号位置。两种功能活性的分析(报告基因 表达)和蛋白质结合(凝胶迁移率改变分析，GMSA) 该地区正在进行中。GMSA数据确认SP1位置 -139.1.6kbp启动子的一部分显示了特异的蛋白质结合(或 在小鼠核提取液中)。为了描述活体内的 这些序列的活性，我们亚克隆了1.6kbp的区域和部分 转移到荧光素酶或β荧光素酶上游的报告载体中- 半乳糖苷酶编码区。初步结果显示，1.6kBP 区域作为一个整体并不促进荧光素酶的表达。要检测 上调该基因的表达，将这些载体导入CR 表达培养的小鼠胚胎脑细胞正在进行中。 其他发现PC-12细胞已经被转染了一种质粒 包含在CMV启动子控制下的CR编码序列。 我们正在研究异位CR表达是否会影响神经元 存活、分化或凋亡。这些细胞正在被观察 神经生长因子诱导分化前后。 最后，我们发现受体、黄体生成素受体和睾酮 在雄性大鼠出生后的前6周内表达一致。卵泡刺激素受体和 β-肌动蛋白以完全不同的平行时间表达 当然了。这可能表明CR与间质细胞有关 大鼠睾酮生成的个体发育和可能的调控 睾丸。

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.