A sensation for inflation: initial swim bladder inflation in larval zebrafish is mediated by the mechanosensory lateral line.

Larval zebrafish achieve neutral buoyancy by swimming up to the surface and taking in air through their mouths to inflate their swim bladders. We define this behavior as 'surfacing'. Little is known about the sensory basis for this underappreciated behavior of larval fish.

A sensation for inflation: initial swim bladder inflation in larval zebrafish is mediated by the mechanosensory lateral line.

Larval zebrafish achieve neutral buoyancy by swimming up to the surface and taking in air through their mouths to inflate their swim bladders. We define this behavior as 'surfacing'. Little is known about the sensory basis for this underappreciated behavior of larval fish.

Teaching Dose-response Relationships Through Aminoglycoside Block of Mechanotransduction Channels in Lateral Line Hair Cells of Larval Zebrafish.

Here we introduce a novel set of laboratory exercises for teaching about hair cell structure and function and dose-response relationships via fluorescence microscopy. Through fluorescent labeling of lateral line hair cells, students assay aminoglycoside block of mechanoelectrical transduction (MET) channels in larval zebrafish. Students acquire and quantify images of hair cells fluorescently labeled with FM 1-43, which enters the hair cell through MET channels.

An improved predictive recognition model for Cys(2)-His(2) zinc finger proteins.

Cys(2)-His(2) zinc finger proteins (ZFPs) are the largest family of transcription factors in higher metazoans. They also represent the most diverse family with regards to the composition of their recognition sequences. Although there are a number of ZFPs with characterized DNA-binding preferences, the specificity of the vast majority of ZFPs is unknown and cannot be directly inferred by homology due to the diversity of recognition residues present within individual fingers.

Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA).

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe context-dependent assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA-generated ZFNs, we rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max.

Predicting success of oligomerized pool engineering (OPEN) for zinc finger target site sequences.

Background: Precise and efficient methods for gene targeting are critical for detailed functional analysis of genomes and regulatory networks and for potentially improving the efficacy and safety of gene therapies. Oligomerized Pool ENgineering (OPEN) is a recently developed method for engineering C2H2 zinc finger proteins (ZFPs) designed to bind specific DNA sequences with high affinity and specificity in vivo. Because generation of ZFPs using OPEN requires considerable effort, a computational method for identifying the sites in any given gene that are most likely to be successfully targeted by this method is desirable.

Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion.

Zinc-finger nucleases (ZFNs) have been successfully used for rational genome engineering in a variety of cell types and organisms. ZFNs consist of a non-specific FokI endonuclease domain and a specific zinc-finger DNA-binding domain. Because the catalytic domain must dimerize to become active, two ZFN subunits are typically assembled at the cleavage site.

Engineering single Cys2His2 zinc finger domains using a bacterial cell-based two-hybrid selection system.

Individual synthetic Cys2His2 zinc finger domains with novel DNA-binding specificities can be identified from large randomized libraries using selection methodologies such as phage display. We have previously demonstrated that a bacterial cell-based two-hybrid system is at least as effective as phage display for selecting zinc fingers with desired specificities from such libraries. In this chapter we provide updated, detailed protocols for performing zinc finger selections using the bacterial two-hybrid system.

Oligomerized pool engineering (OPEN): an 'open-source' protocol for making customized zinc-finger arrays.

Engineered zinc-finger nucleases (ZFNs) form the basis of a broadly applicable method for targeted, efficient modification of eukaryotic genomes. In recent work, we described OPEN (oligomerized pool engineering), an 'open-source,' combinatorial selection-based method for engineering zinc-finger arrays that function well as ZFNs. We have also shown in direct comparisons that the OPEN method has a higher success rate than previously described 'modular-assembly' methods for engineering ZFNs.

Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells.

We report here homologous recombination (HR)-mediated gene targeting of two different genes in human iPS cells (hiPSCs) and human ES cells (hESCs). HR-mediated correction of a chromosomally integrated mutant GFP reporter gene reaches efficiencies of 0.14%-0.

Zinc Finger Database (ZiFDB): a repository for information on C2H2 zinc fingers and engineered zinc-finger arrays.

Zinc fingers are the most abundant DNA-binding motifs encoded by eukaryotic genomes and one of the best understood DNA-recognition domains. Each zinc finger typically binds a 3-nt target sequence, and it is possible to engineer zinc-finger arrays (ZFAs) that recognize extended DNA sequences by linking together individual zinc fingers. Engineered zinc-finger proteins have proven to be valuable tools for gene regulation and genome modification because they target specific sites in a genome.

Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification.

Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays.

Engineering Cys2His2 zinc finger domains using a bacterial cell-based two-hybrid selection system.

Synthetic Cys2His2 zinc finger domains with novel DNA-binding specificities can be identified from large randomized libraries using selection methodologies such as phage display. It has been previously demonstrated that a bacterial cell-based two-hybrid system is at least as effective as phage display for selecting zinc fingers with desired specificities from these libraries. In this chapter the authors provide updated and detailed protocols for performing zinc finger selections using the bacterial two-hybrid system.

DNA-binding Specificity Is a Major Determinant of the Activity and Toxicity of Zinc-finger Nucleases.

The engineering of proteins to manipulate cellular genomes has developed into a promising technology for biomedical research, including gene therapy. In particular, zinc-finger nucleases (ZFNs), which consist of a nonspecific endonuclease domain tethered to a tailored zinc-finger (ZF) DNA-binding domain, have proven invaluable for stimulating homology-directed gene repair in a variety of cell types. However, previous studies demonstrated that ZFNs could be associated with significant cytotoxicity due to cleavage at off-target sites.

DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases.

The engineering of proteins to manipulate cellular genomes has developed into a promising technology for biomedical research, including gene therapy. In particular, zinc-finger nucleases (ZFNs), which consist of a nonspecific endonuclease domain tethered to a tailored zinc-finger (ZF) DNA-binding domain, have proven invaluable for stimulating homology-directed gene repair in a variety of cell types. However, previous studies demonstrated that ZFNs could be associated with significant cytotoxicity due to cleavage at off-target sites.

Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method.

The C2H2 zinc finger is the most commonly utilized framework for engineering DNA-binding domains with novel specificities. Many different selection strategies have been developed to identify individual fingers that possess a particular DNA-binding specificity from a randomized library. In these experiments, each finger is selected in the context of a constant finger framework that ensures the identification of clones with a desired specificity by properly positioning the randomized finger on the DNA template.

Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly.

Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest.