Carroll Dana, Morton J Jason, Beumer Kelly J, Segal David J
Department of Biochemistry, University of Utah School of Medicine, 15 N. Medical Drive East, Room 4100, Salt Lake City, Utah 84112, USA.
Nat Protoc. 2006;1(3):1329-41. doi: 10.1038/nprot.2006.231.
Zinc finger nucleases (ZFNs) are hybrid proteins that have been developed as targetable cleavage reagents for double-stranded DNA, both in vitro and in vivo. This protocol describes the design and construction of new DNA-binding domains comprised of zinc fingers (ZFs) directed at selected DNA sequences. Because the ZFNs must dimerize to cut DNA, they are designed in pairs for any new site. The first step is choosing a DNA segment of interest and searching it for sequences that can be recognized by combinations of existing ZFs. The second step is the construction of coding sequences for the selected ZF sets. Third, these coding sequences are linked to that of the nonspecific cleavage domain from the FokI restriction endonuclease in a cloning vector of choice. Finally, the ZFNs are expressed in Escherichia coli, partially purified, and tested in vitro for cleavage of the target sequences to which they were designed. If all goes smoothly, design, construction and cloning can be completed in about two weeks, with expression and testing completed in one additional week.
锌指核酸酶（ZFNs）是一种杂交蛋白，已被开发为可在体外和体内对双链DNA进行靶向切割的试剂。本方案描述了由针对选定DNA序列的锌指（ZFs）组成的新DNA结合结构域的设计和构建。由于ZFNs必须二聚化才能切割DNA，因此针对任何新位点都要成对设计。第一步是选择感兴趣的DNA片段，并在其中搜索可被现有ZFs组合识别的序列。第二步是构建所选ZF集的编码序列。第三步，将这些编码序列与来自FokI限制性内切核酸酶的非特异性切割结构域的编码序列连接到所选的克隆载体中。最后，在大肠杆菌中表达ZFNs，进行部分纯化，并在体外测试它们对设计靶向的序列的切割能力。如果一切顺利，设计、构建和克隆大约可在两周内完成，表达和测试再用一周完成。
