Chromosomal manipulation by site-specific recombinases and fluorescent protein-based vectors.
Chromosomal manipulation by site-specific recombinases and fluorescent protein-based vectors.
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通过位点特异性重组酶和基于荧光蛋白的载体进行染色体操作。
DOI：
10.1371/journal.pone.0009846
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发表时间：
2010-03-24
期刊：
影响因子：
3.7
通讯作者：
Kinoshita K
中科院分区：
文献类型：
--
作者：
Uemura M;Niwa Y;Kakazu N;Adachi N;Kinoshita K
15年前首次报道了在哺乳动物细胞中进行染色体操作的可行性。虽然这项技术是有用的，在染色体背景下的基因调控的精确理解，有限数量的实验室已经在实际操作中使用它，因为相关的技术困难。为了克服实际的障碍，我们开发了一个铬介导的染色体重组系统，使用荧光蛋白和各种位点特异性重组酶。这些技术使得能够快速构建靶向载体，容易识别染色体重排的细胞，并且重排在连接处留下最少的人工元件。将该系统应用于人类细胞系，我们成功地概括了人类疾病中两种类型的致病性染色体易位：MYC/IgH和BCR/ABL 1。通过诱导靶向同一染色体的两个loxP位点之间的重组，我们可以用不同颜色的荧光标记具有loxP间片段缺失或重复的细胞。此外，我们证明了染色体内重组频率与两个重组位点之间的距离成反比，暗示了该频率作为接近传感器的未来应用。我们的染色体操作方法可用于基因靶向可能的特定细胞类型（例如胚胎干细胞）。该系统的实验性使用将为基因组生物学开辟新的视野，包括建立由易位和拷贝数变异引起的疾病的细胞和动物模型。
Feasibility of chromosomal manipulation in mammalian cells was first reported 15 years ago. Although this technique is useful for precise understanding of gene regulation in the chromosomal context, a limited number of laboratories have used it in actual practice because of associated technical difficulties. To overcome the practical hurdles, we developed a Cre-mediated chromosomal recombination system using fluorescent proteins and various site-specific recombinases. These techniques enabled quick construction of targeting vectors, easy identification of chromosome-rearranged cells, and rearrangement leaving minimum artificial elements at junctions. Applying this system to a human cell line, we successfully recapitulated two types of pathogenic chromosomal translocations in human diseases: MYC/IgH and BCR/ABL1. By inducing recombination between two loxP sites targeted into the same chromosome, we could mark cells harboring deletion or duplication of the inter-loxP segments with different colors of fluorescence. In addition, we demonstrated that the intrachromosomal recombination frequency is inversely proportional to the distance between two recombination sites, implicating a future application of this frequency as a proximity sensor. Our method of chromosomal manipulation can be employed for particular cell types in which gene targeting is possible (e.g. embryonic stem cells). Experimental use of this system would open up new horizons in genome biology, including the establishment of cellular and animal models of diseases caused by translocations and copy-number variations.
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影响因子：
11.4
作者：
Zhang, Y;Wienands, J;Reth, M
通讯作者：
Reth, M
DOI：
10.1073/pnas.91.5.1706
发表时间：
1994-03-01
影响因子：
11.1
作者：
QIN, MM;BAYLEY, C;OW, DW
通讯作者：
OW, DW
DOI：
10.1073/pnas.92.16.7376
发表时间：
1995-08-01
影响因子：
11.1
作者：
VANDEURSEN, J;FORNEROD, M;GROSVELD, G
通讯作者：
GROSVELD, G
影响因子：
5.3
作者：
Zheng, BH;Sage, M;Bradley, A
通讯作者：
Bradley, A
影响因子：
8
作者：
Smith, AJH;Xian, J;Rabbitts, PH
通讯作者：
Rabbitts, PH