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

Abstract

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.

Figure 1

Plasmid map of the SuperZiF plasmids GNN, ANN, and CNN. Unique restriction sites were placed between each zinc-finger module to enable isolation of individual and subsets of zinc fingers. The zinc-finger sequence was synthesized by Blue Heron and cloned into the pUCminusMCS vector. See Supplementary Figure 1 for complete maps and the Supplemenatry Sequence Archive for plasmid sequences.

Figure 2

Library cloning scheme. The SuperZiF vector is digested with XhoI and SpeI and ligated into pCSV (see Supplementary Figure 2 for a complete map and the Supplementary Sequenced Archive for the plasmid sequence). Next, the 1ZF library is cut with XmaI and SpeI to create a 1ZF insert and with AgeI and SpeI to create 1ZF vector (XmaI and AgeI have compatible overhangs and are both destroyed after ligation). These two pieces are ligated to create a 2ZF library. The 2ZF library is cut with AgeI and SpeI to create a 2ZF vector and the 1ZF insert from the previous step is ligated into this vector to create a 3ZF vector. Additional fingers may be added in a successive manner until the desired library length is obtained. This scheme can also be used to create designed clones by using individual fingers at each step rather than the libraries.

Figure 3

Agarose gel electrophoresis (1.5% in 1×TAE with EtBr) of individual clones from the 5ZF GNH/ANN library. Colonies were selected randomly and tested by SfiI enzyme digestion. All clones released a single 500-bp insert after digestion, reflecting the low background (library members with more or less than 5 zinc-fingers) of the library.

Figure 4

Western blot of 4ZF and 5ZF GNH/ANN library expression with detection via an HA tag with anti-HA-peroxidase antibody (Roche). Libraries were cloned into the pMX-IRES-GFP retroviral vectors as both VP64 and KRAB and transduced into MDA-MB-231 cells.