This work was supported by the National Institutes of Health (DP1CA174426 to Carlos F. Barbas) and ShanghaiTech University, Shanghai, China (to J.L). Molecular graphics were generated using PyMol.

1. Cloning
<ol>
	<li>Obtain alanine-substituted two-finger ZiF domains that have been sub-cloned into the pET-28 expression vector system and are available upon request (pET-2F-ZiF).34</li>
	<li>PCR amplify EmGFP from the plasmid Emerald-pBAD with the primers 5&#8217; XmaI-EmGFP (5&#8217;-GGAAATTGCCCGGGATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3&#8217;; XmaI site in bold) and 3&#8217; SacI-EmGFP (5&#8217;-CGGATCTGAGCTCTTACTTGTACAGCTCGTCCATGCCGAG-3&#8217;; SacI site in bold).
	<ol>
		<li>Use 5 ng of template DNA, 10 &#956;l of 10x polymerase buffer, 1 Units (U) of Taq DNA polymerase, 0.2 mM each dNTP and 0.2 &#956;M of each primer in a 100 &#956;l solution with the remaining volume made up of distilled/deionized water. Use the cycling conditions: 95 &#176;C for 5 min; 30 cycles of 95 &#176;C for 30 sec, 55 &#176;C for 30 sec and 72 &#176;C for 1 min; 72 &#176;C for 10 min. Purify the PCR product by gel extraction and determine DNA concentration using a spectrophotometer measuring Abs260 x 50 &#956;g/ml.</li>
	</ol>
	</li>
	<li>Digest pET-2F-ZiF and the insert encoding EmGFP with the restriction enzymes XmaI and SacI in recommended buffer for 3 hr at 37 &#176;C using 10 U of enzyme per 1 &#956;g of DNA. Visualize DNA by agarose gel electrophoresis using a fluorescent intercalating dye, such as ethidium bromide.</li>
	<li>Purify the digested DNA by gel extraction kit and determine DNA concentration by a spectrophotometer measuring Abs260 x 50 &#956;g/ml.</li>
	<li>Ligate the purified EmGFP-encoding DNA into 50-100 ng of pET-2F-ZiF using 1 U of T4 DNA Ligase for at least 1 h at RT. For best results, perform the ligation reaction using a 6:1 insert-to-vector molar ratio.</li>
	<li>Thaw 50 &#956;l of any chemically competent XL-1 Blue Escherichia coli cells on ice and mix gently with 10-20 ng of ligated pET-2F-ZiF-EmGFP.</li>
	<li>Keep on ice for 30 min. Heat shock the mixture at 42 &#176;C for 90 sec and recover the cells in 2 ml of Super Optimal broth with Catabolite repression (SOC) for 1 hr at 37 &#176;C with shaking.</li>
	<li>Spread 100 &#956;l of recovery culture on a lysogeny broth (LB) agar plate with 50 &#956;g/ml kanamycin and incubate O/N at 37 &#176;C.</li>
	<li>The following day, inoculate 6 ml of Super Broth (SB) or LB culture containing 50 &#956;g/ml kanamycin with one colony from the LB agar plate and culture O/N at 37 &#176;C. 
	Note: Colony PCR using the primers 5&#8217; XmaI-EmGFP and 3&#8217; SacI-EmGFP could be used to identify positive clones prior to miniprep.</li>
	<li>Purify pET-2F-ZiF-EmGFP by miniprep and confirm plasmid by DNA sequencing using T7 promoter (5&#8217;-TAATACGACTCACTATAGGG-3&#8217;).</li>
</ol>
2. Expression and Purification
<ol>
	<li>Thaw 50 &#956;l of chemically competent BL21 E. coli cells on ice and mix gently with 100 ng of pET-2F-ZiF-EmGFP plasmid. Transform as described in steps 1.7-1.8.</li>
	<li>The following day, inoculate 10 ml of LB medium containing 50 &#956;g/ml kanamycin with a single colony and grow O/N at 37 &#176;C with shaking.</li>
	<li>The following day, dilute the 10 ml of O/N culture into 1 L of LB medium supplemented with 50 &#956;g/ml kanamycin, 0.2% glucose and 100 &#956;M ZnCl2. Grow the culture at 37 &#176;C with shaking to an optical density at 600 nm (OD600) of 0.8 and induce protein expression with 2 mM isopropyl-&#946;-D-1-thiogalactopyranoside (IPTG). After 6 hr of growth at 37 &#176;C, harvest cells by centrifugation at 5,000 x g for 10 min at 4 &#176;C. 
	Note: Induction conditions are highly variable and depend on the stability of the proteins being expressed. Monitor the OD600 every 30 min until an OD600 of 0.8 is reached.</li>
	<li>Resuspend the cell pellet in 5 ml of lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 100 &#956;M ZnCl2, 1 mM dithiothreitol (DTT), 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 mM imidazole, pH 8.0). Lyse the cells on ice by sonication with the following setting: 50% power output, 4 min process time with 5 sec on/10 sec off intervals. Avoid overheating the solution.</li>
	<li>Centrifuge the cell lysate at 25,000 x g for 30 min at 4 &#176;C and transfer the supernatant into a fresh collection tube. For best results, perform all the following steps at 4 &#176;C.</li>
	<li>Run the supernatant through a column pre-packed with 1 ml of equilibrated Ni-NTA slurry. Wash the resin with 20 ml of wash buffer (50 mM Tris-HCl, 500 mM NaCl, 100 &#956;M ZnCl2, 1 mM DTT, 1 mM MgCl2 and 30 mM imidazole, pH 8.0).</li>
	<li>Elute the protein with 5 ml of elution buffer (50 mM Tris-HCl, 500 mM NaCl, 100 &#956;M ZnCl2, 1 mM DTT, 1 mM MgCl2, and 300 mM imidazole, pH 8.0).</li>
	<li>Buffer exchange the eluted protein with storage buffer (50 mM Tris-HCl, 500 mM NaCl, 100 &#956;M ZnCl2, 1 mM DTT, 1 mM MgCl2 and 10% glycerol, pH 8.0) and concentrate the protein to at least 40 &#956;M using a spin concentrator by centrifugation according to the manufacturer&#8217;s instructions.</li>
	<li>Determine protein concentration by BCA or Bradford assay. Mix 2 &#956;l of purified proteins with 2 &#956;l 2&#215; SDS-PAGE loading dye, boil at 95 &#176;C for 10 min and then resolve on 4%-20% Tris-Glycine SDS-PAGE to assess protein purity (Figure 2).</li>
</ol>
3. Protein Storage
<ol>
	<li>Aliquot the concentrated protein, flash freeze in liquid nitrogen and store at -80 &#176;C. For EmGFP fusion proteins, cover the tube with aluminum foil in order to protect the protein from light.</li>
	<li>Avoid repeated freezing and thawing of the protein solution. Note: ZiF-fused proteins are stable under these conditions for at least 1 month. Inappropriate or &#62;4 month storage may lead to protein precipitation or photobleaching of EmGFP.</li>
</ol>
4. Protein Transduction
<ol>
	<li>Maintain HeLa cells in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) and 1% antibiotic-antimycotic at 37 &#176;C in a fully humidified atmosphere with 5% CO2. 
	Note: Cells passaged more than 30 times are not recommended for protein transduction.</li>
	<li>Pre-coat a 24-well plate with 500 &#956;l of 50 &#956;g/ml of poly-lysine for 30 to 60 min at 25 &#176;C. Seed cells onto a 24-well plate at a density of 2 &#215; 105 cells per well. At 24 hr after seeding, or once cells are between 80%-90% confluent, remove media from each well and wash with 500 &#956;l of pre-warmed serum-free DMEM (SFM).</li>
	<li>To each well, add 250 &#956;l of SFM containing 2 &#956;M of ZiF-EmGFP protein and 100 &#956;M ZnCl2. Incubate cells at 37 &#176;C for 1 hr. Note: ZiF domains enter cells primarily through macropinocytosis,34 which is an energy-dependent mechanism. Therefore, cells must be incubated at 37 &#176;C for efficient protein internalization.</li>
	<li>Remove media from cells and wash three times with 500 &#956;l of calcium- and magnesium-free Dulbecco&#8217;s phosphate-buffered saline (DPBS) supplemented with 0.5 mg/ml of heparin. Note: Heparin is necessary to remove surface-bound protein that might complicate downstream analyses.</li>
	<li>(Optional) Repeat treatments up to three times for enhanced delivery.</li>
	<li>Isolate treated cells immediately after heparin wash by digestion with 0.05% trypsin-EDTA at 37 &#176;C for 2 min. Re-suspend detached cells in 0.5 ml of DPBS supplemented with 1% FBS.</li>
	<li>Measure the fluorescence intensity of each sample by flow cytometry35 using the fluorescein isothiocyanate (FITC) channel (Figure 3). Adjust the forward scatter (FSC) and side scatter (SSC) to place the population of interest on scale, ensuring that populations with different properties are resolved from each other.</li>
	<li>Collect 10,000 live events for each sample and analyze the data using flow cytometry data analysis software.36 Normalize fluorescence from HeLa cells treated with ZiF-EmGFP to those cells treated only with SFM.</li>
</ol>

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The authors have nothing to disclose.

Here, a step-by-step protocol for protein delivery using cell-permeable zinc-finger (ZiF) domains is presented. The ZiF domain does not reduce the activity of fused enzymatic cargo34; allows for the production and purification of proteins in yields nearly identical to those observed with unmodified protein; and can transport proteins and enzymes into a wide range of cell types with efficiencies that exceed traditional cell-penetrating peptide or protein transduction domain systems. Together, these findings ind...

Highly efficient and versatile protein delivery strategies are critical for many basic research and therapeutic applications. The direct delivery of purified proteins into cells represents one of the safest and easiest methods for achieving this.1,2 Unlike strategies that rely on gene expression from nucleic acids,3-5 protein delivery poses no risk of insertional mutagenesis, is independent of the cellular transcription/translation machinery and allows for an immediate effect. However, the lack of simple and generalizable methods for endowing cell-penetrating activity onto proteins routinely confounds their direct entry into cells. Current methods for facilitating intracellular protein delivery are based on the use of naturally occurring6-8 or designed cell-penetrating peptides,9-12 supercharged transduction domains,13,14 nanoparticles15 and liposomes,16 virus-like particles17,18 and polymeric microsphere materials.19 Unfortunately, many of these approaches are hampered by low cellular uptake rates,20,21 poor stability,22 inadvertent cell-type specificity,23 low endosomal escape properties24 and toxicity.25 In addition, many protein transduction technologies reduce the bioactivity of the delivered proteins.14
Our laboratory previously demonstrated that zinc-finger nuclease (ZFN) proteins &#8212; chimeric restriction endonucleases consisting of a programmable Cys2-His2 zinc-finger DNA-binding protein and the cleavage domain of the FokI restriction endonuclease26-28&#160;&#8212; are inherently cell-permeable.29 This surprising cell-penetrating activity was shown to be an intrinsic property of the custom-designed zinc-finger domain, a DNA-binding platform that has emerged as a powerful tool for targeted genome engineering,30-32 and considered to be the result of the constellation of six positively charged residues on the protein surface. Indeed, several DNA-binding proteins, including c-Jun and N-DEK have been shown to possess an innate capacity to cross cell membranes.33 More recently, our laboratory expanded on these results and demonstrated that the cell-penetrating activity of zinc-finger (ZiF) domains could be leveraged for intracellular protein delivery. Genetic fusion of either one- or two-finger ZiF domains to specific protein cargo led to uptake efficiencies that exceeded many conventional cell-penetrating peptide delivery systems.34 Most notably, ZiF-mediated delivery did not compromise the activity of fused enzymatic cargo and facilitated high levels of cytosolic delivery. Collectively, these findings demonstrate the potential of the ZiF domain for facilitating the efficient and facile delivery of proteins, and potentially more diverse types of macromolecules, into cells.
Here, a detailed step-by-step protocol on how to implement ZiF technology for protein delivery in mammalian cells is presented. We previously constructed a suite of one-, two-, three-, four-, five- and six-finger ZiF domains that lack the ability to bind DNA, due to substitution of each of the &#945;-helical DNA-binding residues, but are capable of delivering proteins into cells34 (Figure 1). The production and transduction of Emerald GFP (EmGFP) into HeLa cells using a two-finger ZiF domain is described. This protocol is extensible to almost any protein capable of soluble expression in Escherichia coli and nearly any mammalian cell type. Expected results are provided and strategies for maximizing the performance of this system are also discussed.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>XmaI</td>
			<td>New England Biolabs</td>
			<td>R0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI</td>
			<td>New England Biolabs</td>
			<td>R0156L</td>
			<td></td>
		</tr>
		<tr>
			<td>Expand High Fidelity PCR system</td>
			<td>Roche</td>
			<td>11759078001</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td></td>
		</tr>
		<tr>
			<td>4-20% Tris-Glycine Mini protein gels, 1.5 mm, 10 wells</td>
			<td>Life Technologies</td>
			<td>EC6028BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Laemmli Sample Buffer</td>
			<td>BioRad</td>
			<td>161-0737</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Life Technologies</td>
			<td>15224-017</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) Competent E. coli</td>
			<td>New England Biolabs</td>
			<td>C2527I</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Scientific</td>
			<td>R0391</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>208086-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher Scientific</td>
			<td>PR-V3151&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Thermo Scientific</td>
			<td>36978</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose Resin</td>
			<td>QIAGEN</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Units</td>
			<td>EMO Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11966-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Flat Bottom Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, No Calcium, No Magnesium</td>
			<td>Life Technologies</td>
			<td>21600010</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparan Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>H4777</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano Drop ND-1000 spectrophotometer&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Two-finger ZiF-EmGFP fusion proteins can be expressed in E. coli with &#62;95% homogeneity and high yields (&#62;25 mg/ml) (Figure 2). In general, one- and two-finger ZiF fusion proteins can be produced in quantities nearly identical to those of wild-type unmodified protein. However, in some contexts, five- and six-finger ZiF fusion proteins are unable to be produced in yields high enough for downstream applications.
Direct application of two-finger ZiF-EmGFP protein ...

Watch this Scientific Journal Video about Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains at JoVE.com

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.

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 ...

direct-protein-delivery-to-mammalian-cells-using-cell-permeable-cys2

Research

JoVE Journal

Biology

9.8K Views.  The Scripps Research Institute. 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.

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

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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.

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

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

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 ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...