Modular system for the construction of zinc-finger libraries and proteins.
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.
Gonzalez
Beatriz
B
The Skaggs Institute for Chemical Biology, La Jolla, California, USA.
Schwimmer
Lauren J
LJ
Fuller
Roberta P
RP
Ye
Yongjun
Y
Asawapornmongkol
Lily
L
Barbas
Carlos F
CF
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D016335:Zinc Fingers / Q000235:genetics*
D019151:Peptide Library; D011994:Recombinant Proteins; D014157:Transcription Factors