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

为了探讨类固醇合成急性调节蛋白（StAR）在类固醇合成细胞中的结构与功能，本研究确定了小鼠星星基因在类固醇合成细胞中表达的调控元件。首先，我们确定了一个细菌人工染色体（BAC）克隆，其中包括47 kb的转录起始密码子上游，整个小鼠星星结构基因，和62 kb的终止密码子下游的基因。为了检测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多聚腺苷酸化信号的盒插入到BAC克隆的正常起始甲硫氨酸处。产生具有超螺旋BAC DNA的cohi、独立的转基因系。星星/eGFP转基因在整个肾上腺皮质、睾丸的间质细胞以及卵巢的卵泡膜和间质细胞中以高水平表达。免疫组织化学分析，无论是对小鼠星星蛋白的多克隆抗体或单克隆抗体对eGFP蛋白证实了eGFP的肾上腺和性腺的类固醇生成细胞的表达。我们在其他组织中没有观察到eGFP的荧光或免疫活性，包括脑、胸腺、心脏和肾脏。这些结果表明，包括转录起始密码子上游47 kb、整个小鼠星星结构基因和该基因终止密码子下游62 kb的顺式元件足以使星星基因在肾上腺和性腺中表达，并且星星/eGFP BAC转基因小鼠为星星基因的内源表达提供了eGFP谱系标记。

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.