Stable and unstable transgene integration sites in the human genome: extinction of the Green Fluorescent Protein transgene in K562 cells
Stable and unstable transgene integration sites in the human genome: extinction of the Green Fluorescent Protein transgene in K562 cells
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DOI：
10.1016/s0378-1119(00)00353-x
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发表时间：
2000-10-03
期刊：
影响因子：
3.5
通讯作者：
Tuan, D
中科院分区：
文献类型：
--
作者：
Migliaccio, AR;Bengra, C;Tuan, D
在包括基因治疗研究在内的基因转移实验中，整合的转基因在宿主细胞中的表达通常随时间而下降。这种现象的分子基础还不清楚。我们已经使用了绿色荧光蛋白（GFP）基因作为选择标记和报告基因，研究长期转基因整合和表达在K562细胞。用含有与人β-珠蛋白基因座控制区（LCR）的HS 2或HS 3增强子或巨细胞病毒（CMV）增强子偶联的GFP基因的质粒转染的细胞通过单独的荧光激活细胞分选（FACS）或FAGS结合基于共整合的耐药基因的药物选择来分选。随后将两组选定的细胞培养长达250代细胞。在这两组细胞中长期GFP转基因整合和表达的比较揭示了K562基因组包含两种类型的转基因整合位点：i）大量的不稳定位点，其允许转录但不允许转基因的长期整合，从而在60-250代细胞中消除转基因，和ii）罕见的稳定位点，其允许有效转录和长期整合。转基因的长期稳定整合至少200代细胞。我们的研究结果表明，随着时间的推移，绿色荧光蛋白表达的灭绝是由于至少部分消除的基因从宿主基因组中，而不是完全转录沉默的基因。然而，转基因的长期、稳定表达可以在含有整合到罕见的、稳定的宿主位点中的转基因的细胞中实现。(C)2000 Elsevier Science B. V.保留所有权利。
In gene transfer experiments including gene therapy studies, expression of the integrated transgenes in host cells often declines with time. The molecular basis of this phenomenon is not clearly understood. We have used the Green Fluorescent Protein (GFP) gene as both a selectable marker and a reporter to study long-term transgene integration and expression in K562 cells. Cells transfected with plasmids containing the GFP gene coupled to the HS2 or HS3 enhancer of the human beta -globin Locus Control Region (LCR) or the cytomegalovirus (CMV) enhancer were sorted by either fluorescence-activated-cell-sorting (FACS) alone or FAGS combined with drug selection based on a co-integrated drug resistance gene. The two groups of selected cells were subsequently cultured for long periods up to 250 cell generations. Comparison of long-term GFP transgene integration and expression in these two groups of cells revealed that the K562 genome contains two types of transgene integration sites: i) abundant unstable sites that permit transcription but not long-term integration of the transgenes and thus eliminate the transgenes in 60-250 cell generations and ii) rare stable sites that permit both efficient transcription and long-term stable integration of the transgenes for at least 200 cell generations. Our results indicate that extinction of GFP expression with time is due at least in part to elimination of the gene from the host genome and not entirely to transcriptional silencing of the gene. However, long-term, stable expression of the transgene can be achieved in cells containing the transgene integrated into the rare, stable host sites. (C) 2000 Elsevier Science B.V. All rights reserved.