Sabatino D E, Wong C, Cline A P, Pyle L, Garrett L J, Gallagher P G, Bodine D M
Hematopoiesis Section, Genetics and Molecular Biology Branch, NHGRI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2000 Sep 15;275(37):28549-54. doi: 10.1074/jbc.M004043200.
In red blood cells ankyrin (ANK-1) provides the primary linkage between the erythrocyte membrane skeleton and the plasma membrane. We have previously demonstrated that a 271-bp 5'-flanking region of the ANK-1 gene has promoter activity in erythroid, but not non-erythroid, cell lines. To determine whether the ankyrin promoter could direct erythroid-specific expression in vivo, we analyzed transgenic mice containing the ankyrin promoter fused to the human (A)gamma-globin gene. Sixteen of 17 lines expressed the transgene in erythroid cells indicating nearly position-independent expression. We also observed a significant correlation between the level of Ank/(A)gamma-globin mRNA and transgene copy number. The level of Ank/(A)gamma mRNA averaged 11% of mouse alpha-globin mRNA per gene copy at all developmental stages. The addition of the HS2 enhancer from the beta-globin locus control region to the Ank/(A)gamma-globin transgene resulted in Ank/(A)gamma-globin mRNA expression in embryonic and fetal erythroid cells in six of eight lines but resulted in absent or dramatically reduced levels of Ank/(A)gamma-globin mRNA in adult erythroid cells in eight of eight transgenic lines. These data indicate that the minimal ankyrin promoter contains all sequences necessary and sufficient for erythroid-specific, copy number-dependent, position-independent expression of the human (A)gamma-globin gene.
在红细胞中，锚蛋白（ANK - 1）提供了红细胞膜骨架与质膜之间的主要连接。我们之前已经证明，ANK - 1基因的一个271bp的5'侧翼区域在红系细胞系而非非红系细胞系中具有启动子活性。为了确定锚蛋白启动子是否能在体内指导红系特异性表达，我们分析了含有与人类（A）γ - 珠蛋白基因融合的锚蛋白启动子的转基因小鼠。17个品系中的16个在红系细胞中表达转基因，表明几乎不依赖整合位置的表达。我们还观察到Ank/（A）γ - 珠蛋白mRNA水平与转基因拷贝数之间存在显著相关性。在所有发育阶段，每个基因拷贝的Ank/（A）γ mRNA水平平均为小鼠α - 珠蛋白mRNA的11%。将来自β - 珠蛋白基因座控制区的HS2增强子添加到Ank/（A）γ - 珠蛋白转基因中，导致8个品系中的6个在胚胎和胎儿红系细胞中表达Ank/（A）γ - 珠蛋白mRNA，但在8个转基因品系中的8个成年红系细胞中导致Ank/（A）γ - 珠蛋白mRNA水平缺失或显著降低。这些数据表明，最小的锚蛋白启动子包含了人类（A）γ - 珠蛋白基因红系特异性、拷贝数依赖性、整合位置非依赖性表达所必需且足够的所有序列。
