Sequences governing RNA splicing are difficult to study in situ due to the great difficulty of traditional targeted mutagenesis. Zinc-finger nuclease (ZFN) technology allows for the rapid and efficient introduction of site-specific mutations into mammalian chromosomes. Using a ZFN pair along with a donor plasmid to manipulate the outcomes of DNA repair, we introduced several discrete, targeted ...

The generation of specific mutant animal models is critical for functional analysis of human genes. The conventional gene targeting approach in embryonic stem cells (ESCs) by homologous recombination is however laborious, slow, expensive, and limited to species with functional ESCs. It is therefore a long-sought goal to develop an efficient and simple alternative gene targeting strategy. Here w...

Introduction Zinc Finger Nuclease (ZFN) technology has provided researchers with a tool for integrating exogenous sequences into most cell lines or genomes in a precise manner. Using current methods, the efficiency of targeted integration (TI) into the host genome is generally low and is highly dependent on the ZFN activity at the genomic locus of interest. It is unknown if the ZFN binding and ...

Recent studies have shown that zinc finger nucleases (ZFNs) are powerful reagents for making site-specific genomic modifications. The generic structure of these enzymes includes a ZF DNA-binding domain and nuclease domain (Fn) are separated by an amino acid "linker" and cut genomic DNA at sites that have a generic structure (site1)-(spacer)-(site2) where the "spacer" separates the two binding s...

Designer nucleases, like zinc-finger nucleases (ZFNs), represent valuable tools for targeted genome editing. Here, we took advantage of the gamma-retroviral life cycle and produced vectors to transfer ZFNs in the form of protein, mRNA and episomal DNA. Transfer efficacy and ZFN activity were assessed in quantitative proof-of-concept experiments in a human cell line and in mouse embryonic stem c...

Zinc finger nucleases (ZFNs) have been used successfully to create genome-specific double-strand breaks and thereby stimulate gene targeting by several thousand fold. ZFNs are chimeric proteins composed of a specific DNA-binding domain linked to a non-specific DNA-cleavage domain. By changing key residues in the recognition helix of the specific DNA-binding domain, one can alter the ZFN binding...

Childhood lead poisoning is a costly and largely preventable public health problem that lowers IQs, decreases attention spans, and leads to the development of other childhood intellectual disabilities. Furthermore, recent evidence links developmental lead poisoning with the etiology of disorders that appear much later in life, such as Alzheimer's disease, Parkinson's disease, and schizophrenia....

The Wilms' tumour suppressor gene WT1 encodes a protein involved in urogenital development and disease. The salient feature of WT1 is the presence of four 'Krüppel'-type C(2)-H(2) zinc fingers in the C-terminus. Uniquely to WT1, an evolutionarily conserved alternative splicing event inserts three amino acids (KTS) between the third and fourth zinc fingers, which disrupts DNA binding. The ratio ...

Zinc-finger nucleases (ZFNs) have been used for genome engineering in a wide variety of organisms; however, it remains challenging to design effective ZFNs for many genomic sequences using publicly available zinc-finger modules. This limitation is in part because of potential finger-finger incompatibility generated on assembly of modules into zinc-finger arrays (ZFAs). Herein, we describe the v...