Modular system for the construction of zinc-finger libraries and proteins

被引：65

作者：

Gonzalez, Beatriz ^{[1
,2
,3
]}

Schwimmer, Lauren J. ^{[1
,2
,3
]}

Fuller, Roberta P. ^{[1
,2
,3
]}

Ye, Yongjun ^{[1
,2
,3
]}

Asawapornmongkol, Lily ^{[1
,2
,3
]}

Barbas, Carlos F., III ^{[1
,2
,3
]}

机构：

[1] Scripps Res Inst, Skaggs Inst Chem Biol, La Jolla, CA 92037 USA

[2] Scripps Res Inst, Dept Mol Biol, La Jolla, CA 92037 USA

[3] Scripps Res Inst, Dept Chem, La Jolla, CA 92037 USA

来源：

NATURE PROTOCOLS | 2010年 / 5卷 / 04期

基金：

美国国家卫生研究院;

关键词：

ARTIFICIAL TRANSCRIPTION FACTORS; CONTROLLING GENE-EXPRESSION; DNA-BINDING PROTEINS; DOUBLE-STRANDED DNA; GROWTH-FACTOR-A; HUMAN-CELLS; IN-VIVO; HOMOLOGOUS RECOMBINATION; COMBINATORIAL LIBRARIES; PHENOTYPIC ALTERATION;

D O I：

10.1038/nprot.2010.34

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Engineered zinc-finger transcription factors (ZF-TF) are powerful tools to modulate the expression of specific genes. Complex libraries of ZF-TF can be delivered into cells to scan the genome for genes responsible for a particular phenotype or to select the most effective ZF-TF to regulate an individual gene. In both cases, the construction of highly representative and unbiased libraries is critical. In this protocol, we describe a user-friendly ZF technology suitable for the creation of complex libraries and the construction of customized ZF-TFs. The new technology described here simplifies the building of ZF libraries, avoids PCR-introduced bias and ensures equal representation of every module. We also describe the construction of a customized ZF-TF that can be transferred to a number of expression vectors. This protocol can be completed in 9-11 d.

引用

页码：791 / 810

页数：20

共 69 条

[51] Chimeric nucleases stimulate gene targeting in human cells [J].