A minigene containing four discrete cis elements recapitulates GATA-1 gene expression in vivo
A minigene containing four discrete cis elements recapitulates GATA-1 gene expression in vivo
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DOI：
10.1046/j.1365-2443.2002.00595.x
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发表时间：
2002-12-01
期刊：
影响因子：
2.1
通讯作者：
Yamamoto, M
中科院分区：
文献类型：
--
作者：
Ohneda, K;Shimizu, R;Yamamoto, M
背景资料：条柱ATA-1下的（G）条柱ATA-1下的（h）条柱造血增强子下的（e）条柱造血增强子下的（G1HE），位于造血第一外显子5 '端3.9和2.6 kb之间，是红系细胞中加塔-1基因转录所必需的。然而，G1 HE是不足以赋予加塔-1基因在体内的组织特异性，表明额外的调控序列是necessary.Results：我们在这里证明，其他两个上游启动子元件包含一个双加塔基序或两个CACCC盒也是必不可少的报告基因在红系细胞中的转基因小鼠的表达。这三个顺式作用区的组合足以在原始红系细胞中进行报告基因表达，如通过将元件连接在一起形成659 bp人工（GdC）小基因所证明的。小基因激活的报告基因从内源性或外源性胸苷激酶启动子的转录，保留细胞类型特异性。在GdC小基因的第一个内含子中添加320 bp片段可维持报告基因在定型阶段的表达。此外，一个线的转基因小鼠，表达加塔-1 cDNA的控制下，完整的979 bp的minigene拯救加塔-1生殖系突变小鼠胚胎lethality.Conclusions：一个组合的四个不同的序列基序合作作为一个基本的功能单元加塔-1红细胞在体内转录。
Background: The (G) under bar ATA-(1) under bar (h) under bar aematopoietic (e) under bar nhancer (G1HE), located between 3.9 and 2.6 kb 5' to the haematopoietic first exon, is essential for GATA-1 gene transcription in erythroid cells. However, G1HE is not sufficient to confer tissue specificity on the GATA-1 gene in vivo , indicating that additional regulatory sequences are necessary.Results: We demonstrate here that two other upstream promoter elements containing a double GATA motif or two CACCC boxes are also indispensable for reporter gene expression in erythroid cells in the transgenic mouse. The combination of these three cis -acting regions was sufficient for reporter expression in primitive erythroid cells, as demonstrated by linking the elements together into a 659 bp artificial (GdC) minigene. The minigene activated the transcription of a reporter gene from either the endogenous or an exogenous thymidine kinase promoter, retaining cell type-specificity. The addition of a 320 bp fragment in the first intron to the GdC minigene sustained reporter expression in the definitive stage. Moreover, a line of transgenic mouse that expressed GATA-1 cDNA under the control of the complete 979 bp minigene rescued GATA-1 germ line mutant mice from embryonic lethality.Conclusions: A combination of four distinct sequence motifs co-operatively serve as a fundamental functional unit for GATA-1 erythroid transcription in vivo .