StAR gene structure-function analysis using immortalized adrenal steroidogenic cell line

本研究的目的是在体外研究小鼠类固醇生成急性调节蛋白（星星）基因在类固醇生成细胞系中的结构与功能关系。一个来自野生型小鼠（野生细胞系）和另一个来自星星敲除小鼠（星星（-）细胞系），2.采用磷酸钙法将野生型星星基因转染星星（-）细胞株。3.通过诱变获得了5个突变的星星基因;（1）一个没有线粒体靶向序列，（2）一个没有最后28个氨基酸，这是由于星星基因突变而在人类疾病中最常见的相同突变体，（3）一个具有S57 A（4）一个用S196 A（另一个推定的磷酸化位点从丝氨酸被替换为丙氨酸），和（5）一个用S57 A和S196 A两者。将突变的StAR基因导入表达载体中，用磷酸钙法将突变的StAR基因导入星星（-）细胞系中，在确认突变的StAR基因表达后，测定StAR（-）细胞系条件培养液中的肾上腺类固醇激素，这些研究将有助于揭示星星基因结构中的关键功能位点。

The aim of this study was to elucidate the structure-function relationship of mouse steroidogenic acute regulatory protein (StAR) gene in steroidogenic cell line in vitro.1.We established two types of immortalized adrenal steroidogenic cell lines ; one from wild type mice (wild cell line) and the other from StAR knockout mice (StAR (-) cell line), using temperature sensitive SV40 large T antigen transgenic mice.2.We transfected wild type StAR gene into StAR (-) cell line by calcium phosphate method. We then confirmed the expression of wild StAR mRNA by RT-PCR.3.We generated five mutated StAR genes by mutagenesis ; (1)one without mitochondrial targeting sequence, (2)one without last 28 amino acid, which is the identical mutant most commonly found in human disease due to StAR gene mutation, (3)one with S57A (putative phosphorylation site was substituted from serine to alamine), (4)one with S196A (another putative phosphorylation site was substituted from serine to alamine), and (5)one with both S57A and S196A. Each mutated StAR gene was introduced into expression vector.4.We are transfecting these mutant StAR gene into Star (-) cell line by calcium phosphate method.5.After confirming the expression of each mutated StAR gene, we will measure the adrenal steroid hormone in conditioned media from StAR (-) cell line, into which either wild type or each mutant StAR gene was transfected.These studies will highlight the pivotal functional sites in StAR gene structure.