Development of Mammalian Cell Lines with lac Operator-Tagged Chromosomes.
Development of Mammalian Cell Lines with lac Operator-Tagged Chromosomes.
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具有 lac 操作标记染色体的哺乳动物细胞系的开发。
DOI：
10.1101/pdb.prot4903
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Belmont,AndrewS
中科院分区：
文献类型：
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作者：
Strukov,YuriG;Belmont,AndrewS
发现和使用荧光蛋白来标记染色体蛋白已经产生了基本的结构信息，以及对动力学的洞察，这在以前是无法获得的。本协议描述了一种使用lac操纵子/阻遏子系统标记特定染色体位置的方法，其中细菌操纵子序列的直接重复与识别这些序列的绿色荧光蛋白(GFP)标记的蛋白质偶联。通过定向克隆产生直接的lac操作符重复。虽然使用直接重复序列，而不是反向重复序列，减少了高拷贝数细菌宿主内的重组，但即使是直接重复序列也是不稳定的，需要使用特殊的细菌宿主和低拷贝数质粒进行克隆。用传统的转化方法将lac基因重复序列引入真核细胞。该协议描述了分离具有不同转基因拷贝数的稳定细胞克隆的技术，以及用于目视筛选大量稳定细胞克隆的几种方法，以分离包含具有所需特征的标记染色体区域的稀有克隆。
The discovery and use of fluorescent proteins to label chromosomal proteins has yielded basic structural information as well as insights into dynamics that were previously inaccessible. This protocol describes a method for tagging specific chromosome sites using the lac operator/repressor system, wherein direct repeats of bacterial operator sequences are coupled with green fluorescent protein (GFP)-tagged proteins that recognize these sequences. Direct lac operator repeats are generated by directional cloning. Although the use of direct repeats, as opposed to inverted repeats, reduces recombination within the bacterial host at high copy number, even the direct repeats are unstable, requiring the use of special bacterial hosts and low-copy-number plasmids for cloning. The introduction of the lac operator repeats into eukaryotic cells uses traditional transformation methods. Techniques for the isolation of stable cell clones with varying transgene copy numbers are described in the protocol, as are several methods for visual screening of large numbers of stable cell clones to isolate rare clones containing labeled chromosomal regions with desired features.