Name: a two-step strategy that combines epigenetic modification and biomechanical cues to generate mammalian pluripotent cells
Uploaded: 2020-08-29T13:00:00
Description: Watch this Scientific Journal Video about A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells at JoVE.com

This work was funded by Carraresi Foundation and MiND FoodS Hub ID: 1176436. All the authors are members&#160;of the COST Action CA16119 In vitro 3-D total cell guidance and fitness (CellFit).

All studies were reviewed and approved by the Ethical Committee of the University of Milan. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH). Human cells isolation from healthy adult individuals was approved by the Ethical Committee of the Ospedale Maggiore Policlinico, Milano. All the methods in our study were carried out in accordance with the approved guidelines.
1. Skin fibro...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-azacytidine+affects+TET2+and+histone+transcription+and+reshapes+morphology+of+human+skin+fibroblasts.">5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts.</a> Scientific Reports. 6, 37017 (2016).</li><li>Pennarossa, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+erasing+and+pancreatic+differentiation+of+dermal+fibroblasts+into+insulin-producing+cells+are+boosted+by+the+use+of+low-stiffness+substrate.">Epigenetic erasing and pancreatic differentiation of dermal fibroblasts into insulin-producing cells are boosted by the use of low-stiffness substrate.</a> Stem Cell reviews and reports. 14 (3), 398-411 (2018).</li><li>Pennarossa, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+PTFE+micro-bioreactor+to+promote+3D+cell+rearrangement+and+maintain+high+plasticity+in+epigenetically+erased+fibroblasts.">Use of a PTFE micro-bioreactor to promote 3D cell rearrangement and maintain high plasticity in epigenetically erased fibroblasts.</a> Stem Cell Reviews and Reports. 15 (1), 82-92 (2019).</li><li>Constantinides, P. 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During the last decades, several studies focused on the development of strategies to revert a terminally differentiated cell towards a less committed and higher permissive state. The protocol here described allow the generation and long-term maintenance of pluripotent cells starting from adult mature terminally differentiated cells. The method combines two independent steps that involve the induction of a high permissive state which is achieved through chemical epigenetic erasing and its subsequent maintenance ensured us...

During the last decades, the widely accepted concept of unidirectional progression towards cell commitment and differentiation was completely revised. It has been demonstrated that cell specification can be reversed, and a terminally differentiated cell can be pushed towards a less committed and higher permissive state, using different methods.
Among the several methods proposed, one of the most promising method involves the use of chemical compounds to induce cells into a so called chemically induced pluripotency. The small molecules used in this approach are able to interact and modify the epigenetic signature of an adult mature cell, avoiding the need of any transgenic and/or viral vector1,2,3,4,5,6,7,8,9,10. Numerous studies have recently shown that it is possible to switch cells from one phenotype to another by providing specific biochemical and biological stimuli that induce the reactivation of hypermethylated genes11,12,13,14,15. These demethylating events allow for the conversion of terminally differentiated cells into a primitive progenitor, a multipotent or a high plasticity/pluripotent cell1,2,3,4,5,6,7,8,9,10.
In parallel, many studies have been recently focussing on the understanding of mechanosensing-related cues and, more specifically, on the possibility to use mechanical forces to directly influence cell plasticity and/or differentiation16,17,18,19. Indeed, it has been clearly demonstrated that the extracellular matrix (ECM) plays a key role in the control of cell fate. In particular, the biomechanical and biophysical signals produced by ECM directly regulate molecular mechanisms and signaling pathways, influencing cell behavior and functions20,21. These recent data have paved the way to the development of novel 3D culture systems that more closely mimic the in vivo cell microenvironment, replicating mechanical and physical stimuli driving cell behaviour.
We here describe a two-step protocol that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. In the first step, cells are incubated with the demethylating molecule 5-aza-cytidine (5-aza-CR). This agent is able to induce a significant global DNA demethylation through a combined effect of the direct ten-eleven translocation 2 (TET2)-mediated action8,10 and the indirect inhibition of the DNA methyltransferases (DNMT)22,23. This step induces the removal of the epigenetic blocks with a subsequent re-activation of pluripotency-related gene expression and, therefore, the generation of high plasticity cells1,2,3,8,10, hereinafter referred as &#8220;epigenetically erased cells&#8221;. In the second step, cells are encapsulated in a 3D culture system. To this end, the non-reactive hydrophobic synthetic compound polytetrafluoroethylene (PTFE; with particle size of 1 &#956;m) is used as micro-bioreactor, that permits the creation of a cellular microenvironment unachievable through the use of traditional 2D culture systems10. The PTFE powder particles adhere to the surface of the liquid drop in which cells are re-suspended and isolate the liquid core from the supporting surface, while allowing gas exchange between the interior liquid and the surrounding environment24. The &#8220;PTFE micro-bioreactor&#8221; thus obtained, also known as &#8220;Liquid Marble&#8221;, encourages cells to freely interact with each other, promoting 3D cell rearrangement25,26,27, and extends and stably maintains the acquired high plasticity state though bio-mechanosensing-related cues10.

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M7522</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>5-Azacytidine</td>
			<td>Sigma-Aldrich</td>
			<td>A2385</td>
			<td>5-aza-CR, for fibroblast epigenetic erasing</td>
		</tr>
		<tr>
			<td>Adenosine</td>
			<td>Sigma-Aldrich</td>
			<td>A4036</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic Solution (100&#215;)</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>Component of fibroblast and ESC media</td>
		</tr>
		<tr>
			<td>CFX96 Real-Time PCR</td>
			<td>Bio-Rad Laboratories</td>
			<td>NA</td>
			<td>Thermal cycler for quantitative PCR</td>
		</tr>
		<tr>
			<td>Cytidine</td>
			<td>Sigma-Aldrich</td>
			<td>C4654</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>41966052</td>
			<td>For fibroblast isolation and culture medium</td>
		</tr>
		<tr>
			<td>DMEM, low glucose, pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>31885023</td>
			<td>For ESC medium</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D5652</td>
			<td>PBS; for biopsy and cell wash and for solution preparation</td>
		</tr>
		<tr>
			<td>Dynabeads mRNA DIRECT Micro Purification Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>61021</td>
			<td>mRNA estraction</td>
		</tr>
		<tr>
			<td>ESGRO Recombinant Mouse LIF Protein</td>
			<td>Sigma-Aldrich</td>
			<td>ESG1106</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated</td>
			<td>Thermo Fisher Scientific</td>
			<td>10500064</td>
			<td>Component of fibroblast and ESC media</td>
		</tr>
		<tr>
			<td>FGF-Basic (AA10-155) Recombinant Human Protein</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHG0024</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890</td>
			<td>For dish coating</td>
		</tr>
		<tr>
			<td>GeneAmp PCR System 2700</td>
			<td>Applied Biosystems</td>
			<td>NA</td>
			<td>Thermal cycler for qualitative PCR</td>
		</tr>
		<tr>
			<td>Global DNA Methylation ELISA Kit</td>
			<td>CELL BIOLABS</td>
			<td>STA-380</td>
			<td>Methylation study</td>
		</tr>
		<tr>
			<td>GoTaq G2 Flexi DNA Polymerase</td>
			<td>Promega</td>
			<td>M7801</td>
			<td>Qualitative PCR</td>
		</tr>
		<tr>
			<td>Guanosine</td>
			<td>Sigma-Aldrich</td>
			<td>G6264</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-10 Nutrient Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>31550031</td>
			<td>For ESC medium</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828028</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>KOVA glasstic slide 10 with grids</td>
			<td>Hycor Biomedical</td>
			<td>87144</td>
			<td>For cell counting</td>
		</tr>
		<tr>
			<td>Leica MZ APO Stereo Microscope</td>
			<td>Leica</td>
			<td>NA</td>
			<td>For organoid observation</td>
		</tr>
		<tr>
			<td>L-Glutamine solution</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td>Component of fibroblast and ESC media</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140035</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>Millex-GS 0.22 &#181;m pore filters</td>
			<td>Millipore</td>
			<td>SLGS033SB</td>
			<td>For solution sterilization</td>
		</tr>
		<tr>
			<td>M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant</td>
			<td>Promega</td>
			<td>M3681</td>
			<td>mRNA reverse transcription</td>
		</tr>
		<tr>
			<td>Multiskan FC Microplate Photometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>51119000</td>
			<td>For ELISA plate reading</td>
		</tr>
		<tr>
			<td>Nikon Eclipse TE300 Inverted Phase Contrast Microscope</td>
			<td>Nikon</td>
			<td>NA</td>
			<td>For cell observation</td>
		</tr>
		<tr>
			<td>Perkin Elmer Thermal Cycler 480</td>
			<td>Perkin Elmer</td>
			<td>NA</td>
			<td>Thermal cycler for reverse transcription</td>
		</tr>
		<tr>
			<td>Poly(tetrafluoroethylene) 1 &#956;m particle size</td>
			<td>Sigma-Aldrich</td>
			<td>430935</td>
			<td>For generating micro-bioreactor</td>
		</tr>
		<tr>
			<td>PureLink Genomic DNA Mini Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K182001</td>
			<td>Genomic DNA estraction</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Cells-to-CT Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM1728</td>
			<td>Quantitative PCR</td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>Sigma-Aldrich</td>
			<td>T1895</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 100X20 mm, Standard</td>
			<td>Sarstedt</td>
			<td>833902</td>
			<td>For fibroblast isolation</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 35X10 mm, Standard</td>
			<td>Sarstedt</td>
			<td>833900</td>
			<td>For Fibroblast isolation</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 35X10 mm, Suspension</td>
			<td>Sarstedt</td>
			<td>833900500</td>
			<td>Bacteriology petri dish for liquid marble culture</td>
		</tr>
		<tr>
			<td>Tissue Culture Plate 96 Well,Standard,F</td>
			<td>Sarstedt</td>
			<td>833924005</td>
			<td>For liquid marble culture</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T3924</td>
			<td>For fibroblast dissociation</td>
		</tr>
		<tr>
			<td>Tube 15ml, 120x17mm, PS</td>
			<td>Sarstedt</td>
			<td>62553041</td>
			<td>For cell suspension centrifugation</td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3003</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
	</tbody>
</table>

a two-step strategy that combines epigenetic modification and biomechanical cues to generate mammalian pluripotent cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

The present protocol describes all the steps to be performed to generate and stably maintain mammalian pluripotent cells from adult somatic cells. This method has been successful with fibroblasts isolated from different mammalian species, namely mouse, porcine and human. The representative results here reported are obtained from all cell lines, irrespectively of the species of origin.
Morphological analyses show that, after 18 h incubation with the demethylating agent 5-aza-CR, fibroblasts enc...

Watch this Scientific Journal Video about A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells at JoVE.com

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

The protocol shown in this video for the first time combines the use of chemical epigenetic erasing with biomechanical cues to induce and maintain pluripotency in adult terminally differentiated cells. The main strength of this protocol is that it does not require transgenic or viral vectors. Moreover, it is robust, highly reproducible, and flexible.This technique does not require the use of gene transfection, and therefore represents a notable technological advance for translational medicine and cell therapy, while supporting long-term culture of high plasticity cells. Demonstrating the procedure will be Georgia Pennarossa, an assistant professor, and Teresina De Iorio, a PhD student from my laboratory. Begin by preparing fresh one-millimolar 5-aza-CR stock solution.Weigh 2.44 milligrams of 5-aza-CR and dissolve it in 10 milliliters of DMEM with high glucose. Re-suspend the powder by vortexing and sterilize the solution with a 0.22-micrometer filter. Prepare 5-aza-CR working solution by diluting one microliter of the stock solution in one milliliter of fibroblast culture medium.Trypsinize the cells as described in the text manuscript and dislodge them with gentle pipetting. Then collect the cell suspension and transfer it into a conical tube. Count cells using a counting chamber under an optical microscope.Centrifuge the cell suspension at 150 times G for five minutes. Then, remove the supernatant and resuspend the pellet to obtain 40, 000 cells in 30 microliters of fibroblast culture medium supplemented with one-micromolar 5-aza-CR. Fill a 35-millimeter Petri dish with PTFE powder to produce a bed and dispense the 30 microliters of cells onto the powder bed.Gently rotate the Petri dish in a circular motion to ensure that PTFE powder entirely covers the surface of the liquid drop to form a liquid marble microbioreactor. Pick up the liquid marble microbioreactor using a 1000-microliter pipette tip that has been cut to accommodate the diameter of the marble. Plate the microbioreactor onto a clean bacteriology Petri dish to stabilize it.Then, transfer the microbioreactor from the Petri dish into a 96-well plate. Slowly add 100 microliters of media from the margin of the well. The microbioreactor should float on top of the media.Incubate the liquid marble microbioreactor for 18 hours at 37 degrees Celsius in a 5%carbon dioxide incubator. After the incubation, collect the liquid marble microbioreactor using a 1000-microliter pipette tip and place it in a new 35-millimeter bacteriology Petri dish. Use a needle to puncture the liquid marble and break it.Recover the formed spheroids with a 200-microliter pipette tip that has been cut at the edge under a stereomicroscope. To wash off 5-aza-CR residuals, transfer the organoids into a Petri dish containing ESC medium. Prepare a new 35-millimeter Petri dish containing the PTFE powder bed and dispense a single organoid in a 30-microliter droplet of ESC culture medium onto the powder using a 200-microliter cut pipette tip.Gently rotate the dish in a circular motion to form a new liquid marble microbioreactor and transfer it into a well of a 96-well plate. Add 100 microliters of media from the margin of the well to slowly bathe the marble. Culture the liquid marble microbioreactors at 37 degrees Celsius and 5%carbon dioxide.Morphological analyses show that after an 18-hour incubation with the demethylating agent 5-aza-CR, fibroblasts from three mammalian species encapsulated in PTFE microbioreactors aggregated and formed 3D spherical structures with a uniform size geometry. Under 3D conditions, 86.31%of encapsulated cells remarkably modified their phenotype. In contrast, post-5-aza-CR cells cultured in 2D standard conditions were considerably smaller in size, with granulated nuclei and retained a monolayer distribution.The morphological changes were accompanied by the onset of pluripotency-related gene expression, both in 3D and 2D post-5-aza-CR cells. Transcription was observed for POU class five homeobox one, NANOG homeobox, ZFP42 zinc finger protein, and sex-determining region Ybox2, which are absent in untreated fibroblasts. A significant upregulation of the 10-11 translocation 2 epithelial cell adhesion molecule and cadherin 1 genes was also observed in 3D post-5-aza-CR cells compared to cells cultured in 2D standard plastic dishes.In parallel, downregulation of the fibroblast-specific marker Thy1 cell surface antigen was detected in both 3D and 2D post-5-aza-CR cells. A significant decrease of methylation levels in both 3D and 2D post-5-aza-CR cells was confirmed with ELISA. DNA methylation levels were significantly lower in 3D post-5-aza-CR cells compared to the cells cultured in 2D conditions.Furthermore, 3D post-5-aza-CR cells retained the acquired 3D spherical structure, high expression levels of pluripotency-related genes, and low DNA methylation levels for 28 days, at which point culture was arrested. The microbioreactors can also be used to maintain optimal conditions for progenitor cell cultures, as well as to boost terminal differentiation efficiency.

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Research

JoVE Journal

Biology

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

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 ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.

Methods for mapping in vivo protein-DNA interactions are becoming crucial for every aspect of genomic research but they are laborious, costly, and time consuming. Here a commercially available robotic liquid handling system that automates chromatin immunoprecipitation for mapping in vivo protein-DNA interactions with limited amounts of cells is presented.

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq ...