1. Custom ZFN Design Process
<ol>
 <li>To expedite the ZFN design process, an investigator should provide:
 <ul>
 <li>Information on the target gene such as the gene name, species, and gene annotation/identification number. </li>
 <li>A clearly stated over-all objective of the experiment (e.g., Knockout or Knockin) </li>
 <li>Any specific factors of the gene target which include atypical gene structure, special biology implicated in the genetic locus, or any homologous regions elsewhere in the genome.</li>
 <li> The specific range of DNA sequence for the zinc finger nucleases to target. </li>
 </ul></li>
 <li> The Sigma bioinformatics team conducts in silico ZFN design to develop initial zinc finger target sites. </li>
 <li>Initial ZFN target sites are provided to a ZFN scientific technical consultant. For more technically complex projects, this Sigma scientist works with the investigators to develop the project strategy. </li>
 <li> The Sigma bioinformatics team conducts an analysis with ZFN algorithms to generate in silico ZFN designs. This includes a whole genome search for off-target sites, repeat masking, and SNP analysis for each potential ZFN target site. </li>
 <li> Bioinformatics provides the top ZFN target sites to the investigator for review. Upon approval of the potential ZFN target sites, this information is provided to the ZFN Production team to begin the ZFN manufacturing process. </li>
</ol>
2. Custom ZFN Production
<ol>
 <li>The Sigma archive of zinc finger modules is used to assemble the approved ZFN designs. </li>
 <li>Each ZFN design is assembled and sequence verified on a high throughput cloning platform. </li>
 <li> Once production of all ZFN designs is complete every ZFN is sent to validation. </li>
</ol>

3. Validation of Custom ZFNs
<ol>
 <li>ZFN projects in human, mouse, rat or CHO species are validated in well characterized cell lines for each species, a yeast based assay is used for other organisms or cell types. </li>
 <li>ZFN constructs are delivered into the appropriate cell line by nucleofection and the ZFNs are expressed. </li>
 <li>DNA is harvested from the pool of ZFN transfected cells and the region of interest is PCR amplified. </li>
 <li> The Cel-1 assay is then conducted on the PCR product. Gel electrophoresis is run on the Cel-1 assay to validated ZFN activity. </li>
 <li>The highest activity ZFN design as confirmed by the Cel-1 assay is then provided to the customer. ZFNs are delivered in mRNA and plasmid DNA along with PCR primers for the Cel-1 assay and genomic DNA as a positive control. </li>
</ol>
4. Delivery of Validated ZFNs by Nucleofection
<ol>
 <li> Seed the cells at a density of 2x105 cells/ml the day before nucleofection. </li>
 <li> On the day of nucleofection, take out Cell Line Nucleofector Kit V and let warm to room temperature.</li>
 <li> Add the supplement to the Nucleofection Solution V according to the manufacturer's protocol. </li>
 <li> Count the cells. Cell density should be between 2.5-5x105 cells/ml. </li>
 <li> Fill a 6-well plate with 2ml of medium in each well and pre-warm in a CO2 incubator at 37 &deg;C for at least 20 minutes prior to nucleofection. </li>
 <li> Centrifuge 2x106 cells per transfection (8x106 total) at 200xg for 5 minutes. </li>
 <li> Wash cells twice with 20 ml of Hank's Balanced Salt Solution (HBSS). </li>
 <li> Prepare experimental tubes: (see table in Custom ZFN technical bulletin) </li>
 <li>Remove the 6-well plate containing media from step (6.5) from incubator. </li>
 <li>Resuspend cells in 400 &mu;l (100 &mu;l/reaction) of Nucleofection Solution V. </li>
 <li> One reaction at a time, add 100 &mu;l of cells to each DNA or mRNA-containing tube. Transfer the mixture to a 2 mm electroporation cuvette and nucleofect on a Nucleofector with the appropriate program. </li>
 <li> Immediately after nucleofection of each sample, use a transfer pipette to add ~500 &mu;l of prewarmed medium from the 6-well plate in step (6.9) to the cuvette. Then, carefully transfer cells from the cuvette to the remaining prewarmed medium in the 6-well plate. </li>
 <li> Finish all reactions and return the 6-well plate to the CO2 incubator at 37 &deg;C. </li>
</ol>
5. Harvesting Genomic DNA after Delivery of ZFNs
<ol>
 <li> 6-well plate &ndash; do not harvest all of the pooled cells. It is important to maintain the culture in order to have cells to single cell dilution clone after you confirm that you have Cel-1 digestion products. One to three days after nucleofection collect the cells to prepare chromosomal DNA using the GenElute Mammalian Genomic DNA Miniprep Kit. </li>
</ol>
6. Cel-1 Assay
<ol>
 <li>PCR amplify the genomic DNA from the ZFN transfected samples and the positive control genomic DNA provided in the kit, using the supplied primers. The PCR reaction is set up with the following conditions: (see table in Custom ZFN technical bulletin).</li>
 <li> To generate heteroduplexes from the PCR homoduplexes take 10 &mu;l of PCR reaction from each ZFN treated sample plus the control and use the following program on a thermocycler: 
 95 &deg;C, 10 minutes
 
 95 &deg;C to 85 &deg;C, -2 &deg;C/second
 
 85 &deg;C to 25 &deg;C, -0.1 &deg;C/second 
4 &deg;C, indefinitely </li>
 <li>Add 1 &mu;l of enhancer and 1 &mu;l of Nuclease S (from Transgenomic Catalog Number 706025) to each reaction and incubate at 42 &deg;C for 20-40 minutes.</li>
 <li> Run the digestions on a 10% PAGE-TBE gel with proper markers, such as DirectLoad WideRange DNA Ladder (Catalog Number D7058) (see results in Custom ZFN technical bulletin). </li>
 <li> Once positive signal has been detected in ZFN-treated populations by cel-I assay, proceed to clonal isolation and characterization. </li>
</ol>
7. Clonal Isolation and Characterization
<ol>
 <li> Single-cell clone the ZFN-treated samples by standard methods including FACS and dilution cloning. </li>
 <li>Allow the clones to expand sufficiently, then split some proportion of cells for genomic DNA harvest, freezing back the remainder as banked material.</li>
 <li> Amplify genomic DNA from the clones using the Cel-I primers and analyze the amplicon by Cel-I assay (with and without WT DNA spiked in), or alternatively use this amplicon to proceed directly to genotyping. </li>
 <li>Genotype candidate clones identified as having edited alleles by the preferred method. </li>
</ol>
8. Representative Results
An example of the entire CompoZr ZFN workflow is presented in Figure 2. ZFNs are delivered to cells where they bind and cleave the appropriate DNA sequence which creates a double-stranded break (DSB). The natural repair process, non-homologous end joining (NHEJ), repairs the DSB. In some instances aberrant NHEJ results in deletion, insertion or substitution of nucleotides. PCR amplification of harvested genomic DNA results in heteroduplex formation between wild type and modified amplicons after the denaturation/annealing step of the PCR reaction. Addition of the Cel-1 enzyme results in cleavage of any heteroduplex molecules. Cel-1 results are resolved by PAGE analysis to confirm ZFN cleavage. Figure 3 provides a schematic of the Cel-1 assay and the expected results.
Representative Cel-1 results are contained in Figures 4 and 5. Figure 4 contains results from a very active pair of ZFNs (21%) in K562 cells; Figure 5 contains results from a less active pair (2.4%) of ZFNs in K562 cells. ZFN activity is confirmed in both figures by the presence of two PCR fragments below the parental PCR fragment.
<img src="/files/ftp_upload/3304/3304fig1.jpg" alt="Figure 1" /> 
Figure 1. Representation of bound zinc finger nucleases. ZFNs are engineered proteins comprised of a zinc finger DNA-binding domain fused to the cleavage domain of the FokI restriction endonuclease. When bound as a heterodimer, ZFNs create a double-stranded break at a user specified DNA sequence.
<img src="/files/ftp_upload/3304/3304fig2.jpg" alt="Figure 2" /> 
Figure 2. Schematic of the Custom ZFN Service Workflow. Graphic representation of the workflow to generate a genetically modified cell line using CompoZr ZFNs.
<img src="/files/ftp_upload/3304/3304fig3.jpg" alt="Figure 3" /> 
Figure 3. Schematic of the Cel-1 assay and results. A) ZFN plasmid or mRNA is delivered to cells. B) Expressed ZFNs bind and cut their target sequence creating a double stranded break (DSB) in a portion of cells. C) Aberrant repair of some DSBs by non-homologous end joining results in nucleotide insertion or deletion. D) Genomic DNA is harvested from the transfected pool of cells and amplified at the locus of interest. E-F) PCR product is denatured and re-annealed creating heteroduplex formation between wild type and modified amplicons. G) The Cel-1 mismatch endonuclease assay results in cleavage of heteroduplex molecules. H) Cel-1 enzyme digests are resolved by PAGE. The observed ratio of cleavage product to parenteral band, determined by ImageJ software, indicates the fraction cut and efficiency of the ZFNs.
<img src="/files/ftp_upload/3304/3304fig4.jpg" alt="Figure 4" /> 
Figure 4. Cel-1 results from 21% active ZFNs. ImageJ software is available at: <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>.
<img src="/files/ftp_upload/3304/3304fig5.jpg" alt="Figure 5" /> 
Figure 5. Cel-1 results from 2.4% active ZFNs. ImageJ software is available at: <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>.

<ol>
	<li>Kim, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+restriction+enzymes:+zinc+finger+fusions+to+FokI+cleavage+domain.">Hybrid restriction enzymes: zinc finger fusions to FokI cleavage domain.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 1156-1160 (1996).</li><li>Urnov, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+endogenous+human+gene+correction+using+designed+zinc-finger+nucleases.">Highly efficient endogenous human gene correction using designed zinc-finger nucleases.</a> Nature. 435, 646-651 (2005).</li><li>Moehle, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+addition+into+a+specified+location+in+the+human+genome+using+designed+zinc+finger+nucleases.">Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 3055-3060 (2007).</li><li>Meyer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+targeting+by+homologous+recombination+in+mouse+zygotes+mediated+by+zinc-finger+nucleases.">Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 15022 (2010).</li><li>Geurts, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockout+rats+via+microinjection+of+zinc-finger+nucleases.">Knockout rats via microinjection of zinc-finger nucleases.</a> Science. 325, 433 (2009).</li><li>Meng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+inactivation+in+zebrafish+using+engineered+zinc-finger+nucleases.">Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases.</a> Nat. Biotechnol. 26, 695-701 (2008).</li><li>Young, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeted+gene+disruption+in+the+soma+and+germ+line+of+the+frog+Xenopus+tropicalis+using+engineered+zinc-finger+nucleases.">Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases.</a> Proc. Natl. Acad. Sci. U.S.A. , (2011).</li><li>Wanatabe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockout+of+exogenous+EGFP+gene+in+porcine+somatic+cells+using+zinc-finger+nucleases.">Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases.</a> Biochem. Biophys. Res. Commun. 402, 14-18 (2010).</li><li>Santiago, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+knockout+in+mammalian+cells+by+using+engineered+zinc-finger+nucleases.">Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 5809-5814 (2008).</li><li>Lui, P. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+triple-gene+knockout+mammalian+cell+line+using+engineered+zinc-finger+nucleases.">Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases.</a> Biotechnol. Bioeng. 106, 97-105 (2010).</li><li>Moehle, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+addition+into+a+specified+location+in+the+human+genome+using+designed+zinc+finger+nucleases.">Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 3055-3060 (2007).</li><li>Doyon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+cold+shock+enhances+zinc-finger+nuclease+mediated+gene+disruption.">Transient cold shock enhances zinc-finger nuclease mediated gene disruption.</a> Nat. Methods. 7, 459-460 (2010).</li></ol>

Researchers can provide ZFN modified cells or animals to collaborators under the standard Research License Agreement. In this circumstance Sigma asks that a material transfer agreement is put in place between Sigma, the ZFN customer and their collaborator. The research license may be viewed at: <a href="http://www.sigma.com/zfn" target="_blank">http://www.sigma.com/zfn</a>.
If there is a potential for a commercial application using ZFN modified cells Sigma then asks that a Commercial license is put in place.
Production and Free Access to this article is sponsored by Sigma, Inc.

Once ZFN-modified cells are isolated they have permanent and heritable DNA modifications. This results in the ability to generate stably modified cell lines or breed animals with desired genetic modifications to conduct research. CompoZr Custom ZFNs provide a robust method for genome editing in a wide variety of cell types. Publication of successful genome editing with ZFNs includes but isn't limited to human cell lines, mouse, rat, zebrafish, frog, porcine and CHO research models.3,4,5,6,7,8,9 The ability to create a double-stranded break at the desired target site in the specified cell type is assured when the ZFNs are properly delivered into a cell. It's also possible to perform sequential zinc finger modifications to a cell in order to have more than one genetic modification within a cell.10 The ability to select a specific target sequence and only modify that particular locus is a primary cause for the ability to create multiple modifications.
The CompoZr Custom ZFN certificate of analysis is generated with the very reagents provided in the kit thus assuring that all components of the kit have been thoroughly validated for customer success. Critical steps of the workflow from beginning to end are presented below:
Cell cloning
The ability to isolate and expand a given cell line should be tested before beginning any ZFN experiments. Single cells should be isolated and expanded to ensure that a clonally-derived population is possible. Some cell lines are recalcitrant in this matter, and tricks such as enrichment for edited clones, or culture with conditioned media can help to overcome these challenges.
Delivery efficiency
Optimization of delivery efficiency is a must before delivery of the ZFNs. Many methods may be used including lipid-based transfection, electroporation, and nucleofection. The ideal delivery method consists of one that affords the most optimal blend of delivery efficiency and cell survival. Quantitating these efficiencies may be estimated by delivering a GFP control plasmid and quantitating by visual inspection or FACS 24-48hrs post-delivery.
Cel-I assay
It is imperative that Cel-I assay is performed in parallel with the appropriate controls to ensure that PCR, digestion, and electrophoresis parameters will not interfere with the interpretation of ZFN experiment. Each kit includes control genomic DNA that is used to create the Cel-I image in the certificate of analysis. Amplification from the control DNA with the provided primers followed by Cel-I assay will allow the user to compare the results with that provided in the Certificate of Analysis (CofA) image, and isolate any potential inefficiencies in this aspect of an experiment. An appropriate negative control such as a mock, or GFP treated cell sample should also be included to allow elucidation of any non-specific cleavage products.
Qualitative/Quantitative analysis of single-cell clones
After ZFN treatment and single cell cloning, populations of cells may be screened for editing at the locus of interest by a number of methods. Cel-I assay may be conducted on genomic DNA from clones for the purpose of identifying candidate clones for further genotyping. It is important to spike WT PCR amplicon into the sample amplicons at a 1:1 ratio before performing the Cel-I digest as homozygous mutant clones will contain homogeneous molecules, and thus convey a negative Cel-I assay result. An alternative method for screening these candidate clones is to design one PCR primer landing directly on the ZFN target site. Used in conjunction with one of the control primers, a negative assay result indicates loss of the WT sequence. A heterozygous clone containing at least one WT allele will yield PCR signal, but may be segregated from fully WT clones by performing the PCR in the context of SYBR qPCR.
Once candidate clones have been identified by the above methods, they may be genotyped by standard pyrosequencing, deep sequencing, or other sequencing methods. For all sequencing methods, an amplicon created from the clonal genomic DNA should be generated. For traditional pyrosequencing alleles may be segregated by TA-cloning the PCR product, and sequencing the individual colonies. For methods such as deep sequencing this step is not necessary.
Homology Directed Repair (HDR)
This protocol provides the method for ZFN mediated gene knockout via NHEJ. Integration of transgenes may also be conducted by introduction of a donor plasmid with homology arms that flank a ZFN cut site.11 In this scenario, the ZFN created double-stranded break is repaired by HDR instead of NHEJ. HDR directed integration of transgenes may be confirmed using similar methods to the procedures provided throughout this protocol.
Troubleshooting
Delivery of ZFNs
ZFN delivery and expression- delivery may be controlled for by delivery of a GFP or other such plasmid for visualization and/or quantitation. Low expression due to promoter incompatibility with the cell type of interest may be overcome by delivery of ZFNs in mRNA format. Additionally, the cold shock method may be utilized to further increase the efficacy of ZFNs in cells.12 If mRNA is used it is imperative that cells are washed prior to delivery to ensure all serum-derived Rnases have been removed. It is also prudent to thaw the mRNA on ice, and add the it to the cells at the very last moment to minimize any chance for degradation.
Cel-I assay
The foundation of the Cel-I assay is specific and ample amplification of the locus of interest. If non-specific amplification products are observed use standard PCR troubleshooting procedures to increase specificity such as optimization of template amount, primer concentration, and cycling parameters. Perform PAGE analysis as opposed to standard agarose electrophoresis as the latter method does not provide adequate resolution and sensitivity. Cel-I digest conditions may also be optimized if no digested product or excessive smearing is observed. Titration of the amount of Cel-1 nuclease and/or length of digestion time can be titrated to improve these aspects.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Nuclease S (Cel-1 Enzyme)</td>
 <td>Transgenomic</td>
 <td>706025</td>
 </tr>
 <tr>
 <td>Expand High Fidelity PCR System</td>
 <td>Roche</td>
 <td>03 300 242 001</td>
 </tr>
 <tr>
 <td>Cell Line Nucleofector Kit V</td>
 <td>Lonza</td>
 <td>VCA-1003</td>
 </tr>
 <tr>
 <td>GenElute HP Endotoxin-Free Plasmid Maxiprep Kit</td>
 <td>Sigma-Aldrich</td>
 <td>NA0400</td>
 </tr>
 </tbody>
</table>

genome editing with compozr custom zinc finger nucleases (zfns)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Watch this Scientific Journal Video about Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs at JoVE.com

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

genome-editing-with-compozr-custom-zinc-finger-nucleases-zfns

Research

JoVE Journal

Biology

25.2K Views.  Sigma Life Science. The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

Video: Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

A Tactile Automated Passive-Finger Stimulator (TAPS)

Although tactile spatial acuity tests are used in both neuroscience research and clinical assessment, few automated devices exist for delivering controlled spatially structured stimuli to the skin. Consequently, investigators often apply tactile stimuli manually. Manual stimulus application is time consuming, requires great care and concentration on the part of the investigator, and leaves many stimulus parameters uncontrolled. We describe here a computer-controlled tactile stimulus system, the Tactile Automated Passive-finger Stimulator (TAPS), that applies spatially structured stimuli to the skin, controlling for onset velocity, contact force, and contact duration. TAPS is a versatile, programmable system, capable of efficiently conducting a variety of psychophysical procedures. We describe the components of TAPS, and show how TAPS is used to administer a two-interval forced-choice tactile grating orientation test.
Corresponding Author: Daniel Goldreich

A Tactile Automated Passive-Finger Stimulator TAPS

We describe a computer-controlled device for investigating the sense of touch: the Tactile Automated Passive-finger Stimulator (TAPS). We describe the components of TAPS, and show how TAPS is used to administer a two-interval forced-choice tactile grating orientation test.

Although tactile spatial acuity tests are used in both neuroscience research and clinical assessment, few automated devices exist for delivering ...

Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain generates electric fields  - that can be recorded using electroencephalography (EEG)- and their concomitant magnetic fields  - detected by MEG. MEG signals are detected by specialized sensors known as superconducting quantum interference devices (SQUIDs). Superconducting sensors require cooling with liquid helium at -270 &deg;C. They are contained inside a vacumm-insulated helmet called a dewar, which is filled with liquid. SQUIDS are placed in fixed positions inside the helmet dewar in the helium coolant, and a subject's head is placed inside the helmet dewar for MEG measurements. The helmet dewar must be sized to satisfy opposing constraints. Clearly, it must be large enough to fit most or all of the heads in the population that will be studied. However, the helmet must also be small enough to keep most of the SQUID sensors within range of the tiny cerebral fields that they are to measure. Conventional whole-head MEG systems are designed to accommodate more than 90% of adult heads. However adult systems are not well suited for measuring brain function in pre-school chidren whose heads have a radius several cm smaller than adults. The KIT-Macquarie Brain Research Laboratory at Macquarie University uses a MEG system custom sized to fit the heads of pre-school children. This child system has 64 first-order axial gradiometers with a 50 mm baseline[2] and is contained inside a magnetically-shielded room (MSR) together with a conventional adult-sized MEG system [3,4]. There are three main advantages of the customized helmet dewar for studying children. First, the smaller radius of the sensor configuration brings the SQUID sensors into range of the neuromagnetic signals of children's heads. Second, the smaller helmet allows full insertion of a child's head into the dewar. Full insertion is prevented in adult dewar helmets because of the smaller crown to shoulder distance in children. These two factors are fundamental in recording brain activity using MEG because neuromagnetic signals attenuate rapidly with distance. Third, the customized child helmet aids in the symmetric positioning of the head and limits the freedom of movement of the child's head within the dewar. When used with a protocol that aligns the requirements of data collection with the motivational and behavioral capacities of children, these features significantly facilitate setup, positioning, and measurement of MEG signals.

The advent of MEG systems sized for young children opens important new opportunities to study brain development. The new system, together with a protocol that aligns experimental requirements with the capacities of children, can be used to study cognitive and language processes in healthy, awake children aged three to six.

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Performing Custom MicroRNA Microarray Experiments

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1. Complex genomes encode at least hundreds of miRNAs, which primarily inhibit the expression of a vast number of target genes post-transcriptionally 2, 3. miRNAs control a broad range of biological processes 1. In addition, altered miRNA expression has been associated with human diseases such as cancers, and miRNAs may serve as biomarkers for diseases and prognosis 4, 5. It is important, therefore, to understand the expression and functions of miRNAs under many different conditions.
Three major approaches have been employed to profile miRNA expression: real-time PCR, microarray, and deep sequencing. The technique of miRNA microarray has the advantage of being high-throughput, generally less expensive, and most of the experimental and analysis steps can be carried out in a molecular biology laboratory at most universities, medical schools and associated hospitals. Here, we describe a method for performing custom miRNA microarray experiments. A miRNA probe set will be printed on glass slides to produce miRNA microarrays. RNA is isolated using a method or reagent that preserves small RNA species, and then labeled with a fluorescence dye. As a control, reference DNA oligonucleotides corresponding to a subset of miRNAs are also labeled with a different fluorescence dye. The reference DNA will serve to demonstrate the quality of the slide and hybridization and will also be used for data normalization. The RNA and DNA are mixed and hybridized to a microarray slide containing probes for most of the miRNAs in the database. After washing, the slide is scanned to obtain images, and intensities of the individual spots quantified. These raw signals will be further processed and analyzed as the expression data of the corresponding miRNAs. Microarray slides can be stripped and regenerated to reduce the cost of microarrays and to enhance the consistency of microarray experiments. The same principles and procedures are applicable to other types of custom microarray experiments.

A simple procedure of performing custom microRNA microarray experiments is described. The steps include isolating RNA, labeling RNA and reference DNA, hybridizing the samples to microarrays, scanning the microarrays, quantifying and analyzing hybridization signals.

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1.  Complex genomes encode ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs. Towards this end, much recent work has focused on identifying candidate genes for the evolution of traits in a variety of species. However, until recently it has been challenging to functionally validate interesting candidate genes. Recently developed tools for genetic engineering make it possible to manipulate specific genes in a wide range of organisms. Application of this technology in evolutionarily relevant organisms will allow for unprecedented insight into the role of candidate genes in evolution. Astyanax mexicanus (A. mexicanus) is a species of fish with both surface-dwelling and cave-dwelling forms. Multiple independent lines of cave-dwelling forms have evolved from ancestral surface fish, which are interfertile with one another and with surface fish, allowing elucidation of the genetic basis of cave traits. A. mexicanus has been used for a number of evolutionary studies, including linkage analysis to identify candidate genes responsible for a number of traits. Thus, A. mexicanus is an ideal system for the application of genome editing to test the role of candidate genes. Here we report a method for using transcription activator-like effector nucleases (TALENs) to mutate genes in surface A. mexicanus. Genome editing using TALENs in A. mexicanus has been utilized to generate mutations in pigmentation genes. This technique can also be utilized to evaluate the role of candidate genes for a number of other traits that have evolved in cave forms of A. mexicanus.

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish.

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs.  Towards this end, much recent ...

Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a systematic approach combining different methodologies to assess the availability of zinc (Zn) in Atlantic salmon (Salmo salar). Considering that several Zn chemical species can be present in an Atlantic salmon feed, it was hypothesised that Zn availability is influenced by the Zn chemical species present in the feed. Thus, in this study, the first protocol is about how to extract the different Zn chemical species from the feed and to analyze them by a size exclusion chromatography-inductively coupled plasma mass spectroscopy (SEC-ICP-MS) method. Subsequently, an in vitro method was developed to evaluate the solubility of dietary Zn in Atlantic salmon feeds. The third protocol describes the method to study the impact of changing Zn chemical species composition on the uptake of Zn in a fish intestinal epithelial model using a rainbow trout gut cell line (RTgutGC). Together, the findings from the in vitro methods were compared with an in vivo study examining the apparent availability of inorganic and organic sources of Zn supplemented to Atlantic salmon feeds. The results showed that several Zn chemical species can be found in feeds and the efficiency of an organic Zn source depends very much on the amino acid ligand used to chelate Zn. The findings of the in vitro methods had less correlation with that outcome of the in vivo study. Nevertheless, in vitro protocols described in this article provided crucial information regarding Zn availability and its assessment in fish feeds.

This article explains in detail a systematic approach to assess micro-mineral availability in Atlantic salmon. The methodology includes tools and models with increasing biological complexity: (1) chemical speciation analysis, (2) in vitro solubility, (3) uptake studies in cell lines, and (4) in vivo fish studies.&#160;

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a ...