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The wmN1 enhancer region in intron 1 is required for expression of human PLP1
Glia ( IF 5.4 ) Pub Date : 2018-04-23 , DOI: 10.1002/glia.23339
Hamdan Hamdan 1 , Pankaj Patyal 1 , Neriman T. Kockara 1 , Patricia A. Wight 1
Glia ( IF 5.4 ) Pub Date : 2018-04-23 , DOI: 10.1002/glia.23339
Hamdan Hamdan 1 , Pankaj Patyal 1 , Neriman T. Kockara 1 , Patricia A. Wight 1
Affiliation
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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.
中文翻译：
内含子1中的wmN1增强子区域是表达人PLP1所必需的
髓磷脂蛋白脂质蛋白基因（PLP1）编码来自中枢神经系统（CNS）的髓磷脂中存在的最丰富的蛋白质。如改变PLP1剂量的突变所证明，其表达必须严格控制。过度表达（PLP1拷贝数升高）和缺乏表达（PLP1缺失）均会导致人类X连锁遗传病。但是，关于控制人类基因表达的机制知之甚少。为了解决这个问题，产生了利用人类PLP1（hPLP1）序列（近6.2 kb的5'-侧翼DNA到外显子2的前38 bp）的转基因小鼠，以驱动lacZ报告基因盒的表达。熏鲑鱼P位点在hPLP1内含子1的1.5 kb区域附近并入，因为它包含与小鼠wmN1区域同源的序列，以前显示它可以增加最小促进的转基因的表达，与CNS发育的活跃髓鞘期相吻合。用亲本6.2hPLP（+）Z / FL转基因产生了八个转基因品系。所有系均在脑中适当表达了转基因，如在白质区和嗅球中用X-gal染色所证明。用普遍表达的Cre-driver从6.2hPLP（+）Z / FL中去除“ wmN1”区域会导致转基因活性急剧下降。这些结果首次证明了wmN1增强子区域：（1）在hPLP1中起作用; （2）与它的天然启动子协同工作，而不仅仅是基础的异源启动子。（3）高水平的hPLP1基因活性是必需的；（4）在空间和时间上都比最初用mPlp1投影的效果更广。
更新日期：2018-04-23
中文翻译：
内含子1中的wmN1增强子区域是表达人PLP1所必需的
髓磷脂蛋白脂质蛋白基因（PLP1）编码来自中枢神经系统（CNS）的髓磷脂中存在的最丰富的蛋白质。如改变PLP1剂量的突变所证明，其表达必须严格控制。过度表达（PLP1拷贝数升高）和缺乏表达（PLP1缺失）均会导致人类X连锁遗传病。但是，关于控制人类基因表达的机制知之甚少。为了解决这个问题，产生了利用人类PLP1（hPLP1）序列（近6.2 kb的5'-侧翼DNA到外显子2的前38 bp）的转基因小鼠，以驱动lacZ报告基因盒的表达。熏鲑鱼P位点在hPLP1内含子1的1.5 kb区域附近并入，因为它包含与小鼠wmN1区域同源的序列，以前显示它可以增加最小促进的转基因的表达，与CNS发育的活跃髓鞘期相吻合。用亲本6.2hPLP（+）Z / FL转基因产生了八个转基因品系。所有系均在脑中适当表达了转基因，如在白质区和嗅球中用X-gal染色所证明。用普遍表达的Cre-driver从6.2hPLP（+）Z / FL中去除“ wmN1”区域会导致转基因活性急剧下降。这些结果首次证明了wmN1增强子区域：（1）在hPLP1中起作用; （2）与它的天然启动子协同工作，而不仅仅是基础的异源启动子。（3）高水平的hPLP1基因活性是必需的；（4）在空间和时间上都比最初用mPlp1投影的效果更广。
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