Transcriptional behavior of LCR enhancer elements integrated at the same chromosomal locus by recombinase-mediated cassette exchange
Transcriptional behavior of LCR enhancer elements integrated at the same chromosomal locus by recombinase-mediated cassette exchange
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DOI：
10.1182/blood.v90.9.3332
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发表时间：
1997-11-01
期刊：
影响因子：
20.3
通讯作者：
Leboulch, P
中科院分区：
文献类型：
--
作者：
Bouhassira, EE;Westerman, K;Leboulch, P
在哺乳动物细胞中，通过重组酶介导的盒交换(RMCE)实现了在预先选择的染色体位置的转基因的有效整合，这是一种利用Cre重组酶和含有不同间隔区的Lox位点的新方法。我们将RMCE应用于人β-珠蛋白基因位点控制区的研究，方法是在MEL细胞中的同一遗传位点整合由人β-珠蛋白启动子驱动的LacZ基因，该基因由人β-珠蛋白启动子单独连接到HS2和HS3，或与HS4结合。在细胞群体水平和用氯化血红素或DMSO诱导分化前后的单个细胞中的表达研究表明，这些增强子的存在与多样化的表达模式有关。我们能够证明所测试的LCR片段通过控制连接的β-珠蛋白启动子的表达概率和转录的稀有程度来发挥作用，这两个因素也取决于Melc的分化状态和位于顺式基因的第二个转录单元的存在。通过整合到染色体中的RMCE结构进行操作的能力应该有助于创建复杂的、合理设计的人工遗传位点。(C)1997年由美国血液病学会主办。
Efficient integration of transgenes at preselected chromosomal locations was achieved in mammalian cells by recombinase-mediated-cassette-exchange (RMCE), a novel procedure that makes use of the CRE recombinase together with Lox sites bearing different spacer regions, We have applied RMCE to the study of the human beta-globin gene Locus Control Region by integrating at the same genetic locus in MEL cells, a LacZ gene driven by the human beta-globin promoter linked to HS2 and HS3 alone or in combination with HS4. Expression studies at the cell population level and in individual cells before and after induction of differentiation with hemin or DMSO show that the presence of these enhancers is associated with variegated patterns of expression, We were able to show that the LCR fragments tested act by controlling both the probability of expression and the rare of transcription of the linked beta-globin promoter, Both of these factors were also dependent on the state of differentiation of the MELc and on the presence of a second transcription unit located in cis. The ability to manipulate by RMCE constructs integrated into chromosomes should help in the creation of complex, rationally designed, artificial genetic loci. (C) 1997 by The American Society of Hematology.