Targeted expression of enhanced green fluorescent protein to steroidogenic cells expressing steroidogenic acute regulatory protein by bacterial artificial chromosome transgenesis in vivo

通过体内细菌人工染色体转基因向表达类固醇生成急性调节蛋白的类固醇生成细胞靶向表达增强型绿色荧光蛋白

基本信息

批准号：
18591167
负责人：
ISHII Tomohiro
金额：
$ 2.52万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

In order to explore structure-function aspects of steroidogenic acute regulatory protein (StAR) within steroidogenic cells, the present research defined the regulatory elements sufficient for expression of mouse Star gene in steroidogenic cells in vivo. First, we identified a bacterial artificial chromosome (BAC) clone that included 47 kb upstream of the transcription initiation codon, the entire mouse Star structural gene, and 62 kb downstream of the termination codon of the gene. To examine the ability of these genomic sequences to target gene expression correctly, we inserted a cassette encoding enhanced green fluorescent protein (eGFP) and a polyadenylation signal from bovine growth hormone into the BAC clone at the normal initiator methionine, Following BAC Modification by homologous recombination in E. cohi, independent transgenic lines with supercoiled BAC DNA were generated. The Star/eGFP transgene was expressed at high levels throughout the adrenal cortex, in Leydig cells in the testes, and in theca and interstitial cells in the ovary. Immunohistochemical analysis with either polyclonal antibodies against mouse StAR protein or monoclonal antibodies against eGFP protein confirmed the expression of eGFP in steroidogenic cells of the adrenal glands and gonads. We did not observe fluorescence or immunological activity of the eGFP in other tissues, including brain, thymus, heart, and kidney. These results indicate that the cis-element including 47 kb upstream of the transcription initiation codon, the entire mouse Star structural gene, and 62 kb downstream of the termination codon of the gene is sufficient for the expression of the Star gene in adrenal glands and gonads and that the Star/eGFP BAC transgenic mice provide an eGFP lineage marker for the endogenous expression of Star gene.

为了探索类固醇生成细胞内类固醇生成急性调节蛋白（StAR）的结构-功能方面，本研究定义了足以在体内类固醇生成细胞中表达小鼠Star基因的调控元件。首先，我们鉴定了一个细菌人工染色体 (BAC) 克隆，其中包括转录起始密码子上游 47 kb、整个小鼠 Star 结构基因以及该基因终止密码子下游 62 kb。为了检查这些基因组序列正确靶向基因表达的能力，我们将编码增强型绿色荧光蛋白（eGFP）和来自牛生长激素的聚腺苷酸信号的盒插入到正常起始子蛋氨酸处的 BAC 克隆中，然后通过同源重组进行 BAC 修饰在大肠杆菌中，产生了具有超螺旋 BAC DNA 的独立转基因系。 Star/eGFP 转基因在整个肾上腺皮质、睾丸的 Leydig 细胞以及卵巢的卵泡膜和间质细胞中高水平表达。使用针对小鼠 StAR 蛋白的多克隆抗体或针对 eGFP 蛋白的单克隆抗体进行的免疫组织化学分析证实了 eGFP 在肾上腺和性腺的类固醇生成细胞中的表达。我们没有在其他组织（包括脑、胸腺、心脏和肾脏）中观察到 eGFP 的荧光或免疫活性。这些结果表明，包括转录起始密码子上游47kb、整个小鼠Star结构基因和该基因终止密码子下游62kb的顺式元件足以使Star基因在肾上腺和性腺中表达Star/eGFP BAC转基因小鼠为Star基因的内源表达提供了eGFP谱系标记。