Name: analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions
Uploaded: 2016-12-28T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions at JoVE.com

This work was supported by the Natural Sciences and Engineering Council of Canada and the Ontario Ministry of Research and Innovation.

1. Preparations for Cell Culture
<ol>
	<li>Purchase commercially available stocks of human cells. 
	NOTE: This protocol utilizes HCT116 colorectal carcinoma and primary human renal proximal tubular epithelial cells (HRPTEC).</li>
	<li>Make 500 ml medium for culture of HCT116: Dulbecco&#39;s Modified Eagle Medium (DMEM)/High glucose medium supplemented with 7.5% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin (P/S).</li>
	<li>Make 500 ml medium for culture of HRPTEC: Epithelial cell medium supplemented with...

<ol>
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The analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions can allow for the identification of novel oxygen-regulated translation initiation factors. The affinity of these factors for the 5&#39; cap of mRNA or other cap-associated proteins can be measured by the strength of their association to m7GTP-linked Agarose beads. One caveat of this technique is that it measures the cap-binding potential of proteins post-lysis, but it is performed under non-denaturing conditions that...

Translational control is emerging as an equally important step to transcriptional regulation in gene expression, especially during periods of cellular stress1. A focal point of translation control is at the rate-limiting step of initiation where the first steps of protein synthesis involve the binding of the eukaryotic initiation factor 4E (eIF4E) to the 7-methylguanosine (m7GTP) 5&#39; cap of mRNAs2. eIF4E is part of a trimeric complex named eIF4F that includes eIF4A, an RNA helicase, and eIF4G, a scaffolding protein required for the recruitment of other translation factors and the 40S ribosome3. Under normal physiological conditions, the vast majority of mRNAs are translated via a cap-dependent mechanism, but under periods of cellular stress approximately 10% of human mRNAs contain 5&#39; UTRs that could allow cap-independent translation intiation1,4. Cap-dependent translation has been historically synonymous with eIF4F, however, stress-specific variations of eIF4F have become a trending topic5-8.
Various cellular stresses cause eIF4E activity to be repressed via the mammalian target of rapamycin complex 1 (mTORC1). This kinase becomes impaired under stress, which results in the increased activity of one of its targets, the 4E-binding protein (4E-BP). Non-phosphorylated 4E-BP binds to eIF4E and blocks its ability to interact with eIF4G causing the repression of cap-dependent translation9,10. Interestingly, a homolog of eIF4E named eIF4E2 (or 4EHP) has a much lower affinity for 4E-BP11, perhaps allowing it to evade stress-mediated repression. Indeed, initially characterized as a repressor of translation due to its lack of interaction with eIF4G12, eIF4E2 initiates the translation of hundreds of mRNAs that contain RNA hypoxia response elements in their 3&#39; UTR during hypoxic stress6,13. This activation is achieved through interactions with eIF4G3, RNA binding protein motif 4, and the hypoxia inducible factor (HIF) 2&#945; to constitute a hypoxic eIF4F complex, or eIF4FH6,13. As a repressor under normal conditions, eIF4E2 binds with GIGYF2 and ZNF59814. These complexes were, in part, identified through Agarose-linked m7GTP affinity resins. This classic method15 is standard in the field of translation and is the best and most commonly used technique to isolate cap-binding complexes in pull down and in vitro binding assays16-19. As the cap-dependent translation machinery is emerging as flexible and adaptable with inter-changing parts6-8,13, this method is a powerful tool to rapidly identify novel cap-binding proteins involved in the stress response. Furthermore, variations in eIF4F could have broad implications as several eukaryotic model systems appear to use an eIF4E2 homolog for stress responses such as A. thaliana20, S. Pombe21, D. melanogaster22, and C. elegans23.
Evidence suggests that variations in eIF4F may not be strictly limited to stress conditions, but be involved in normal physiology24. The oxygen supply to tissues (at capillary ends) or within tissues (measured via microelectrodes) varies from 2-6% in the brain25, 3-12% in the lungs26, 3.5-6% in the intestine27, 4% in the liver28, 7-12% in the kidney29, 4% in muscle30, and 6-7% in bone marrow31. Cells and mitochondria contain less than 1.3% oxygen32. These values are much closer to hypoxia than the ambient air where cells are routinely cultured. This suggests that what were previously thought of as hypoxia-specific cellular processes may be relevant in a physiological setting. Interestingly, eIF4F and eIF4FH actively participate in the translation initiation of distinct pools or classes of mRNAs in several different human cell lines exposed to physiological oxygen or &#34;physioxia&#34;24. Low oxygen also drives proper fetal development33 and cells generally have higher proliferation rates, longer lifespans, less DNA damage and less general stress responses in physioxia34. Therefore, eIF4FH is likely a key factor in the expression of select genes under physiological conditions.
Here, we provide a protocol to culture cells in fixed physiological oxygen conditions or in a dynamic fluctuating range that is likely more representative of tissue microenvironments. One advantage of this method is that cells are lysed within the hypoxia workstation. It is not often clear how the transition from hypoxic cell culture to cell lysis is performed in other protocols. Cells are often first removed from a small hypoxia incubator before lysis, but this exposure to oxygen could affect biochemical pathways as the cellular response to oxygen is rapid (one or two min)35. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. Exploring the activity and composition of eIF4FH and other variations of eIF4F through the method described here will shed light on the intricate gene expression machineries that cells utilize during physiological conditions or stress responses.

<table border="0" cellpadding="0" cellspacing="0" width="901">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:380px;">&#947;-aminophenyl-m7GTP agarose C10-linked beads</td>
			<td style="width:151px;">Jena Bioscience</td>
			<td style="width:165px;">AC-1555</td>
			<td style="width:205px;">Agarose-linked m7GTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm culture dish</td>
			<td>Corning</td>
			<td>877222</td>
			<td>10-cm culture dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 mm culture dish</td>
			<td>Thermofisher</td>
			<td>130183</td>
			<td>15-cm culture dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AEBSF Hydrochloride</td>
			<td>ACROS Organics</td>
			<td>A0356829</td>
			<td>AEBSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose Beads</td>
			<td>Jena Bioscience&#160;</td>
			<td>AC-0015</td>
			<td>Agarose bead control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenol Blue</td>
			<td>Fisher</td>
			<td>BP112-25</td>
			<td>Component of SDS-PAGE loading buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL Centrifuge Tubes</td>
			<td>FroggaBio</td>
			<td>1210-00S</td>
			<td>Used to centrifuge small volumes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Conical Centrifuge Tubes</td>
			<td>Fisher</td>
			<td>1495970C</td>
			<td>Used in culturing primary cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Defined trypsin inhibitor</td>
			<td>Fisher</td>
			<td>R007100</td>
			<td>DTI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreital</td>
			<td>Fisher</td>
			<td>BP172-25</td>
			<td>DTT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epithelial cell medium (complete kit)</td>
			<td>ScienCell</td>
			<td>4101</td>
			<td>Includes serum and growth factor supplements)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td>Component of SDS-PAGE loading buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mM Guanosine 5&#39;-triphosphate, 1 mL</td>
			<td>Jena Bioscience</td>
			<td>272076-0251M</td>
			<td>GTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCT116 colorectal carcinoma</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>Human cancer cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human renal proximal tubular epithelial cells</td>
			<td>ATCC</td>
			<td>PCS-400-010</td>
			<td>HRPTEC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone DMEM/High Glucose</td>
			<td>GE Life Sciences</td>
			<td>SH30022.01</td>
			<td>Standard media for human cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone Penicillin-Streptomycin solution</td>
			<td>GE Life Sciences</td>
			<td>SV30010</td>
			<td>Antibiotic component of DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">H35 HypOxystation</td>
			<td>Hypoxygen</td>
			<td>N/A</td>
			<td>Hypoxia workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Igepal CA-630</td>
			<td>MP Biomedicals</td>
			<td>2198596</td>
			<td>Detergent component of lysis buffer</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Monopotassium phosphate</td>
			<td>Fisher</td>
			<td>P288-500</td>
			<td>KH2PO4</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Potassium chloride</td>
			<td>Fisher</td>
			<td>P217-500</td>
			<td>KCl</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium chloride</td>
			<td>Fisher</td>
			<td>M33-500</td>
			<td>MgCl2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Fisher</td>
			<td>BP358-10</td>
			<td>NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium fluoride</td>
			<td>Fisher</td>
			<td>5299-100</td>
			<td>NaF (phosphatase inhibitor component of lysis buffer)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Disodium phosphate</td>
			<td>Fisher</td>
			<td>5369-500</td>
			<td>Na2HPO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Grade Fetal Bovine Serum</td>
			<td>Seradigm</td>
			<td>1500-500</td>
			<td>FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail (100 x)</td>
			<td>Cell Signalling</td>
			<td>58715</td>
			<td>Component of lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Dodecyl Sulfate</td>
			<td>Fisher</td>
			<td>BP166-100</td>
			<td>SDS</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sodium Orthovanadate</td>
			<td>Sigma</td>
			<td>56508</td>
			<td>Na3VO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>Fisher</td>
			<td>BP152-5</td>
			<td>Component of buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.05% Trypsin-EDTA (1x)</td>
			<td>Life Technologies</td>
			<td>2500-067</td>
			<td>Trypsin used to detach adherent cells</td>
		</tr>
	</tbody>
</table>

analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Analysis of Cap-binding Ability in Response to Oxygen of eIF4E and eIF4E2 in an m7GTP Affinity Column
Figures 1 and 2 represent western blots of typical m7GTP affinity purification of two major cap-binding proteins in response to oxygen fluctuations in two human cell lines: primary human renal proximal tubular epithelial cells (HRPTEC) in Figure 1 and colorect...

Watch this Scientific Journal Video about Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions at JoVE.com

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

The overall goal of this experiment is to capture cap-binding proteins in conditions of low oxygen. This method utilizes an agarose linked seven methyl-guanosine cap analog to investigate cap-binding factors and their interacting partners. This method will help answer key questions in the field of protein synthesis such as identifying novel oxygen regulated factors involved in cap dependent translation.The main advantage of this technique is that cells are kept in a hypoxia workstation up until the point of lysis. The implications of this technique extend towards cancer therapy because cancer cells within a tumor are exposed to very low levels of oxygen and translational control plays a key role in tumor progression. We first had the idea for this method when we read a recent publication from Corona's colleagues reporting that tissue oxygenation is actually closer to what we call hypoxia rather than the normaxia that cells are routinely cultured in.Begin this procedure will cell culture as described in the text protocol. After counting the cells using a hemocytometer feed 150 millimeter cultured dishes containing 15 milliliters of complete medium to obtain a cell density of 2500 to 5000 cells per square centimeter. Use two 150 millimeter dishes for each cap-binding assay.Place the seeded culture dishes in the incubator at 37 degrees celsius and a five percent carbon dioxide atmosphere. After the cells have incubated at least 24 hours place the cells into a hypoxia workstation for the desired time. Then, set the workstation to the appropriate physioxia.Pre-warm one X phosphate buffered saline, or PBS, and trypsin in a 37 degree celsius water bath for 30 minutes. Aliquot 980 microliters of lysis buffer into a 1.5 microliter centrifuge tube for each cap-binding assay being performed. 10 microliters of 100X protease inhibitor cocktail and 10 microliters of 4-benzenesulfonyl fluoride hydrocloride to the 980 microliters of lysis buffer on ice.Discard the media from each 150 millimeter dish in a waste container within the hypoxia workstation. Then, wash the cells with three to four milliliters of warm one X PBS and discard the liquid. Add one milliliter of warm 05 percent trypsin EDTA to each dish and let the dishes sit for two minutes or until the cells are no longer adhered to the plate.Using a pipette transfer the cells of one plate to a 1.5 milliliter micro-centrifuge tube. Repeat as needed so that each plate of cells is transferred to a separate 1.5 milliliter micro-centrifuge tube. Centrifuge the micro-centrifuge tubes at 6000 times g for 90 seconds to pellet the cells.Following centrifugation aspirate the trypsin using a pipette without disturbing the pellet. Wash the cells with warm one X PBS by gently pipetting 200 microliters of PBS into the tube just above the pellet. Then aspirate the PBS without disturbing the pellet.Next, pipette 500 microliters from the one milliliter prepared lysis buffer solution onto the first cellular pellet. Re-suspend the pellet entirely by pipetting up and down. Combine the re-suspended cells with the second cell pellet and re-suspend the second pellet by pipetting up and down.Repeat this process until all pellets have been combined and re-suspended for each sample. Next, combine the lysate with the remaining 500 microliters of lysis buffer in a 1.5 liter micro-centrifuge tube. Remove the samples from the hypoxia workstation.Lyse the cells using gentle agitation by rotating the samples at four degrees celsisus for 1.5 to two hours. Following incubation, centrifuge the lysed cells at 1200 times g for 15 minutes at four degrees celsius to remove cellular debris. Transfer the lysate to a new 1.5 milliliter micro-centrifuge tube and discard the pellet.Reserve a portion of the supernatant to be used as whole cell lysate input control for western blot analysis. To prepare for the cap-binding assay use scissors to remove the tip of the pipette to facilitate collection of the bead slurry. For each sample transfer 50 microliters of the blank agarose bead control slurry and 50 microliters of the m7GTP agarose C10-linked beads slurry to separate 1.5 milliliter micro-centrifuge tubes.Then, pellet the beads by centrifuging the slurry at 500 times g for 30 seconds. Remove the supernatant carefully and re-suspend the beads in 500 microliters of TBS. After repeating these steps pellet the beads at 500 times g for 30 seconds and remove the supernatant.Next, transfer the supernatant containing the lysate to the 1.5 milliliter micro-centrifuge tube containing the blank agarose beads. Incubate for 10 minutes at four degrees celsius with gentle agitation. After 10 minutes, pellet the blank agarose beads by centrifuging at 500 times g for 30 seconds.Following centrifugation transfer the lysate that doesn't bind to the beads to the 1.5 milliliter micro-centrifuge tube containing the m7GTP agarose C10-linked beads. Wash the blank agarose beads by re-suspending the beads in 500 microliters of lysis buffer, pelleting the beads by centrifuging at 500 time g for 30 seconds, and discarding the supernatant. Repeat this four times for a total of five washes.Re-suspend the blank agarose beads in one X SDS-PAGE sample buffer. And boil the beads for 90 seconds at 95 degrees celsius. Store the sample at 20 degrees celsius for future western blot analysis to observe weather the protein of interest binds non-specifically to the bead.Incubate the lysate with the m7GTP agarose C10-linked beads with gentle agitation for one hour at four degrees celsuis to capture cap-binding proteins. Following agitation, pellet the m7GTP agarose C10-linked beads by centrifuging at 500 times g for 30 seconds. After discarding the supernatant, wash the m7GTP agarose C10-linked beads as before.Re-suspend the beads in 600 microliters of lysis buffer and add GTP to a final concentration of one millimolar. Incubate the m7GTP agarose C10-linked beads plus one millimolar GTP with gentle agitation for one hour at four degrees celsius. This will dissociate proteins that non-specifically interact with m7GTP.Pellet the m7GTP agarose C10-linked beads by centrifuging at 500 time g for 30 seconds. Transfer the supernatant to a new 1.5 milliliter micro-centrifuge tube, and add 200 microliters of four X SDS-PAGE sample buffer. After washing the beads as before, re-suspend the beads in one X SDS-PAGE sample buffer and boil at 95 degrees celsius for 90 seconds.Shown here are representative western blots of a typical m7GTP affinity purification of two major cap-binding proteins in response to oxygen fluctuations in primary human renal proximal tubule epithelial cells. The cell lysates were exposed to eight percent, five percent, three percent, or one percent oxygen for 24 hours. The lanes include 10 percent of the whole cell lysate as the input, a GTP washed to measure the specificity for m7GTP, and the proteins bound to m7GTP beads after the GTP wash.Data from at least three independent experiments were quantified by Image J and expressed as relative density units relative to 10 percent input of the whole cell lysate. Results indicate that human cells cultured under physiological oxygen utilize two cap-binding proteins to recruit distinct mRNAs for translation. After watching this video you should have a good understanding of how to capture cap-binding proteins in low oxygen conditions.While attempting this procedure it's important to remember to keep cells in the hypoxia workstation up until the point of lysis, as oxygen can rapidly alter the biochemical properties of these translation initiation factors. Following this procedure other methods like polysome fractionation can be performed to answer additional questions such as which cap-binding proteins associate with active translation under conditions of low oxygen.

analysis-cap-binding-proteins-human-cells-exposed-to-physiological

Research

JoVE Journal

Genetics

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms remain elusive mainly due to the lack of a good in vivo model for studying BEST1 and its mutations under physiological conditions. BEST1 encodes an ion channel, namely BESTROPHIN1 (BEST1), which functions in retinal pigment epithelium (RPE); however, the extremely limited accessibility to native human RPE cells represents a major challenge for scientific research. This protocol describes how to generate human RPEs bearing BEST1 disease-causing mutations by induced differentiation from human pluripotent stem cells (hPSCs). As hPSCs are self-renewable, this approach allows researchers to have a steady source of hPSC-RPEs for various experimental analyses, such as immunoblotting, immunofluorescence, and patch clamp, and thus provides a very powerful disease-in-a-dish model for BEST1-associated retinal conditions. Notably, this strategy can be applied to study RPE (patho)physiology and other genes of interest natively expressed in RPE.

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Here we present a protocol to differentiate retinal pigment epithelium (RPE) cells from human pluripotent stem cells bearing patient-derived mutations. The mutant cell lines may be used for functional analyses including immunoblotting, immunofluorescence, and patch clamp. This disease-in-a-dish approach circumvents the difficulty of obtaining native human RPE cells.

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...