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

为了探索类固醇生成细胞中类固醇生成急性调节蛋白（steroidogenic acute regulatory protein, StAR）的结构和功能，本研究确定了小鼠体内类固醇生成细胞中足以表达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，该克隆包括转录起始密码子上游47 kb、整个小鼠Star结构基因和该基因终止密码子下游62 kb。为了检验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）和牛生长激素多聚腺苷化信号的卡带插入以正常启动剂甲硫氨酸为起始点的BAC克隆中，在大肠杆菌中通过同源重组对BAC进行修饰，获得了具有超螺旋BAC DNA的独立转基因系。Star/eGFP转基因在肾上腺皮质、睾丸间质细胞以及卵巢的卵膜和间质细胞中均有高水平表达。用小鼠StAR蛋白多克隆抗体或eGFP蛋白单克隆抗体进行免疫组化分析，证实eGFP在肾上腺和性腺的类固醇细胞中表达。我们没有观察到eGFP在其他组织中的荧光或免疫活性，包括脑、胸腺、心脏和肾脏。这些结果表明，转录起始密码子上游47 kb的顺式元件、整个小鼠Star结构基因以及该基因终止密码子下游62 kb的顺式元件足以在肾上腺和性腺中表达Star基因，Star/eGFP BAC转基因小鼠为Star基因的内源性表达提供了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.