Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas.
Glia. 2018 Aug;66(8):1763-1774. doi: 10.1002/glia.23339. Epub 2018 Apr 23.
The myelin proteolipid protein gene (PLP1) encodes the most abundant protein present in myelin from the central nervous system (CNS). Its expression must be tightly controlled as evidenced by mutations that alter PLP1 dosage; both overexpression (elevated PLP1 copy number) and lack thereof (PLP1 deletion) result in X-linked genetic disorders in man. However, not much is known about the mechanisms that govern expression of the human gene. To address this, transgenic mice were generated which utilize human PLP1 (hPLP1) sequences (proximal 6.2 kb of 5'-flanking DNA to the first 38 bp of exon 2) to drive expression of a lacZ reporter cassette. LoxP sites were incorporated around a 1.5-kb section of hPLP1 intron 1 since it contains sequence orthologous to the wmN1 region from mouse which, previously, was shown to augment expression of a minimally-promoted transgene coincident with the active myelination period of CNS development. Eight transgenic lines were generated with the parental, 6.2hPLP(+)Z/FL, transgene. All lines expressed the transgene appropriately in brain as evidenced by staining with X-gal in white matter regions and olfactory bulb. Removal of the "wmN1" region from 6.2hPLP(+)Z/FL with a ubiquitously expressed Cre-driver caused a dramatic reduction in transgene activity. These results demonstrate for the first time that the wmN1 enhancer region: (1) is functional in hPLP1; (2) works in collaboration with its native promoter-not just a basal heterologous promoter; (3) is required for high levels of hPLP1 gene activity; (4) has a broader effect, both spatially and temporally, than originally projected with mPlp1.
髓鞘蛋白脂质蛋白基因（PLP1）编码中枢神经系统（CNS）髓鞘中含量最丰富的蛋白质。其表达必须受到严格控制，这一点可以从改变 PLP1 剂量的突变中得到证明；过表达（增加 PLP1 拷贝数）和缺乏（PLP1 缺失）都会导致人类 X 连锁遗传疾病。然而，对于控制人类基因表达的机制，我们知之甚少。为了解决这个问题，生成了利用人 PLP1（hPLP1）序列（5'侧翼 DNA 的近端 6.2kb 到外显子 2 的前 38bp）驱动 lacZ 报告基因盒表达的转基因小鼠。在 hPLP1 内含子 1 的一个 1.5kb 片段中引入了 LoxP 位点，因为它包含与先前显示在中枢神经系统发育的活跃髓鞘形成期增强最小启动子转基因表达的小鼠 wmN1 区域同源的序列。生成了 8 条含有亲本 6.2hPLP(+)Z/FL 转基因的转基因系。所有系在大脑中均适当表达了转基因，这一点可通过在白质区域和嗅球中用 X-gal 染色来证明。用普遍表达的 Cre 驱动将“wmN1”区域从 6.2hPLP(+)Z/FL 中去除，导致转基因活性显著降低。这些结果首次表明 wmN1 增强子区域：（1）在 hPLP1 中具有功能；（2）与天然启动子协同作用，而不仅仅是基本的异源启动子；（3）是 hPLP1 基因高活性所必需的；（4）具有比最初在 mPlp1 中预测的更广泛的空间和时间效应。
