This work was funded by a grant from the National Institutes of Health (GM070736) to H. F&ouml;lsch. S.F. Ang was supported by an A*STAR Graduate Scholarship award, and R.S. Kang was supported by the Cellular and Molecular Basis of Disease Training Program (GM8061)

1. Isolation of Plasmid DNA
<ol>
	<li>Use a Sigma-Aldrich endotoxin-free maxiprep kit to prepare endotoxin free DNA according to the manufacturer's protocol. This kit works for us, because it reliably removes any endotoxins from DNA preparations. Endotoxins that are injected with the DNA into cell nuclei will lead to cell death.</li>
	<li>Add 100 &mu;l phenol/chlorofom/isoamylalcohol (25:24:1) to the isolated DNA, vortex and spin for 1 min at 13,000 rpm in an Eppendorf microcentrifuge. Transfer the upper water phase into a new tube and add 100 &mu;l chloroform/isoamylalcohol (24:1), vortex and spin as above. Transfer the upper water phase containing the DNA to a new tube. This step is needed to remove any protein from the DNA, which prevents clogging of the microinjection needle.</li>
	<li>Precipitate the DNA by adding Na Acetate (pH 6.0) to a final concentration of 300 mM and 2 X vol 100% ethanol. Incubate at -20&deg;C overnight. Spin DNA for 20 min at 13,000 rpm in an Eppendorf microcentrifuge, wash once with 70% ethanol, and resuspend in 300 &mu;l endotoxin-free water (Sigma-Aldrich).</li>
	<li>Determine DNA concentration. A typical DNA concentration ranges from 1 to 5 &mu;g/&mu;l.</li>
</ol>
2. Culture of MDCK Cells
<ol>
	<li>Split MDCK cells. Count cells and seed 4 X 105 cells onto clear 12-mm Transwell filters (0.4 &mu;m pore size, Corning Costar, 3460). For an experiment containing a mock-injection control and two different mutant Rab proteins you need to seed three filters.</li>
	<li>Culture MDCK cells in MEM medium with 2 mM L-glutamine, 0.1 mg/ml penicillin/streptomycin and 7% (vol/vol) fetal bovine serum (= MEM growth media) at 37&deg;C and 5% CO2.</li>
	<li>Change media in the basolateral chamber every day. This aids the polarization process, because it mimics the situation of epithelial cells in animals.</li>
	<li>2 days post-seeding check in a microscope whether the cells are growing in a closed monolayer. If you cannot detect any holes in the monolayer, perform your experiment on day 3. If there are still holes, perform your experiment on day 4.</li>
</ol>
3. Microinjection Procedure and Post-injection Incubations
<ol>
	<li>On the day of the experiment prepare 5 ml MEM growth media plus 50 mM HEPES in one 60 x 15 mm plate for each filter and place into a 39&deg;C incubator. In addition, set up one well of a 12-well plate with 1 ml MEM growth media plus 50 mM HEPES and 0.1 mg/ml cycloheximide per filter, and place into a 31&deg;C incubator.</li>
	<li>Turn on your microinjection microscope and set its heated stage to 39&deg;C. We use an inverted microscope (Axiovert 200, Carl Zeiss MicroImaging, Inc.) with a heated stage, 10X and 32X objectives, and an Eppendorf Femtojet (Injectman NI2). Lastly, open the nitrogen gas tank that supplies the air table with nitrogen.</li>
	<li>Dilute DNA with filtered water (use 0.2 &mu;m filter) to a final concentration of 0.2 mg/ml in a total volume of 10 - 100 &mu;l. Subsequently, spin DNA in Eppendorf microcentrifuge at 13,000 rpm for 30 min. Remove top portion and place in a new tube. This step insures that when you load your diluted DNA into the microinjection needle you can accurately load "clean" DNA. Your DNA is now ready for microinjection.</li>
	<li>Prepare your cells by taking the first filter out of its culture dish. With a surgical blade (Feather Surgical Blade, stainless steel, No 11), cut out the filter from the filter holder and place into 5 ml, 39&deg;C warm MEM growth media plus 50 mM HEPES (60 x 15 mm plate). Weigh down the filter in the culture plate with the surgical blade you used for cutting it out. Place it on the filter such that the hole of the surgical blade surrounds the middle of the filter. Place your culture plate onto the heated stage of the microscope.</li>
	<li>Load 2-3 &mu;l of your diluted DNA (in this case plasmids encoding VSVGts045-GFP = mock injection control) into a microinjection needle (Femtotip II, Eppendorf, 930000043), twist the protective cover of the needle and let it fall to the floor. Now the needle is ready to be screwed into its holder. To do so, press the menu key of your injectman and make sure the valve is shut down. Screw the needle into its holder, beware not to screw in the needle too tightly as that may lead to breakage. Now, press the menu key again, the applied Pc will prevent the media from being sucked into the needle during the microinjeciton procedure. Finally, tap your joystick to erase stored homing.</li>
	<li>To lower the needle onto the cells, use the 10X objective and bring the needle into the light beam above the liquid. Now focus on the cells, bring the focus up again by turning the focus wheel 180&deg; up, and find the needle. Subsequently, slowly move needle into focus, then bring out of focus again (working towards bringing the cells into focus again), bring down needle into focus again. Repeat until needle touches the surface of the media, at which point all you will see is a halo. When you reach a point at which the cells are in focus, but the needle is still fuzzy (i.e. out of the focal plane) change to the 32X objective and fine coarse settings.</li>
	<li>Set the z-limit by touching the apical membrane with the tip of the needle and subtracting about 10 &mu;m as the nuclei are laying approximately 10 &mu;m underneath the apical membrane.</li>
	<li>After you set the z-limit, find the right injection pressure. Start at 95 PSI. If the pressure is too high, your cells will blow up. If your pressure is too low, you will see a white dot that stays, but nothing else happens. With a successful injection, you will see a phase change without a cell size change. For the injection, bring the needle a couple of microns out of the focal plane as fast as possible. Aim with the needle above the nucleus (i.e. the middle of an individual cell) and press the injection button of your joystick, but do not hold the button down.</li>
	<li>Inject 100-500 cells within the hole of your surgical blade that lies on your cells. When you are done, place your cells with the culture dish and surgical blade into a 39&deg;C incubator, and incubate for 2 h. During this time, VSVGts045-GFP is expressed and secreted into the ER. However, at 39&deg;C, VSVGts045-GFP cannot fold correctly and thus cannot leave the ER. Meanwhile, the small GTPase of interest will accumulate in the cytosol in co-injection experiments.</li>
	<li>After 2 h, place your cells into 1 ml MEM growth media plus 50 mM HEPES and 0.1 mg/ml cycloheximide in a 12-well plate and incubate for 2 h at 31&deg;C. During this chase period, VSVGts045-GFP will fold correctly, leave the ER and is delivered to the cell surface.</li>
	<li>Repeat steps 3.1 - 3.10 for filters two (inject plasmids encoding VSVGts045-GFP and for example V5-tagged Rab13T22N) and three (inject plasmids encoding VSVGts045-GFP and for example V5-tagged Rab13Q67L). Each injection will take about 20 - 60 min. However, it is absolutely crucial to treat every filter the same way throughout the temperature shift incubations and surface staining until you fix the cells. After fixation, you may perform the rest of your staining protocol for all filters at the same time.</li>
</ol>
4. Surface Staining for Immunofluorescence Analysis
Note, in order to avoid bleaching of the GFP signal of VSVGts045-GFP, protect the specimens from light by covering with aluminum foil during all subsequent procedures.
<ol>
	<li>If you wish to perform surface staining, place your cells in a culture dish onto a metal plate on ice and wash your cells 1X with ice cold PBS2+ (PBS [0.2 g/liter KCl, 0.2 g/liter KH2PO4, 8 g/liter NaCl, and 2.17 g/liter Na2HPO4 x 7 H2O] plus 0.1 g/liter CaCl2 and 0.1 g/liter MgCl2 x 6 H2O). Subsequently, place 30 &mu;l of an antibody that recognizes the ectodomain of your protein, in this case the mouse monoclonal antibody TK1 (IgG1, obtained from the late Thomas Kreis), onto clean parafilm placed on the metal plate on ice. Place the filter with your cells upside down onto the drop and add a few drops of antibody onto the backside of the filter. Incubate for 1 h on ice.</li>
	<li>Place cells into a 12-well plate, wash 3X with ice cold PBS2+ (or RT warm PBS2+ without prior surface staining), and fix with 3% paraformaldehyde for 15 min at RT.</li>
	<li>Wash cells once with PBS2+ and leave in PBS2+ for 5 min.</li>
	<li>Incubate cells in blocking/permeabilization buffer (BPB) (2% [wt/vol] BSA, 0.4% [wt/vol] saponin in PBS2+) with 10% [vol/vol] goat serum. Incubate 1 h at RT.</li>
	<li>Dilute primary antibodies to detect the expressed Rab GTPase, in this example anti-V5 (mouse monoclonal antibody IgG2a, Invitrogen), 1:200 in BPB. Spin diluted antibodies in an Eppendorf microcentrifuge for 10 min at 13,000 rpm. Place 30 &mu;l of the antibody solution on clean parafilm placed in a wet chamber. Place the cells on filter upside down onto the antibody drop and incubate for 1 h at RT.</li>
	<li>Place cells back (right side up) into a 12-well plate and wash 5X over 30 min with BPB at RT.</li>
	<li>Dilute appropriate secondary antibodies, in this case goat anti-mouse IgG1 Alexa 594-labeled (for VSVG detection on the surface, Invitrogen), and Cy5-labeled secondary antibodies to recognize your Rab GTPase, in this example goat anti-mouse IgG2a Cy5-labeled (Jackson ImmunoResearch), 1:200 into BPB and spin as above. Place 30 &mu;l antibody solution onto clean parafilm in a wet chamber and place cells on filter upside down onto the antibody drop. Incubate 1 h at RT.</li>
	<li>Repeat step 4.6.</li>
	<li>Dip cells on filter 3X into deionized water and place right side up onto microslides. Add 10-15 &mu;l mount (10% [wt/vol] DABCO, 50% [wt/vol] glycerol in distilled water) on top of the cells. Place 18X18 mm micro cover glass on top and using facial tissues gently press the micro cover glass onto the cells. Seal with nail polish.</li>
	<li>Analyze specimens with a confocal microscope. We used a Microsystem LSM 510, Carl Zeiss MicroImaging, Inc. that was equipped with a 63X water immersion lens.</li>
	<li>For the preparation of figures, adjust and combine images using programs such as Adobe Photoshop and Adobe Illustrator.</li>
</ol>
5. Representative Results
For examples on how the co-expression of small GTPases interferes with VSVG sorting kindly refer to published articles for either apical missorting 3, 4 or inhibition of surface delivery 7.
 <img src="/files/ftp_upload/2645/2645fig1.jpg" alt="Figure 1" /> 
Figure 1. In the mock-injection, i.e. an injection of only the plasmid encoding VSVGts045-GFP, the protein is delivered to the basolateral surface of well-polarized MDCK cells as judged by surface staining (in red, Figure 1 A). Note that not all of the VSVGts045-GFP is delivered to the basolateral membrane during the chase at 31&deg;C as evidenced by the extensive intracellular signal for the total protein (in green, Figures 1 A and B). In cells that are not well-polarized VSVGts045-GFP will be delivered also to the apical membrane (Figure 1 B). If your control specimens look like the cell in Figure 1 B, you cannot trust your data and have to repeat the experiment with better-polarized cells. Scale bars are 5 &mu;m.

<ol>
	<li>Mellman, I., Nelson, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature reviews. 9 (11), 833-833 (2008).</li><li>Rodriguez-Boulan, E., Kreitzer, G., Musch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature reviews. 6 (3), 233-233 (2005).</li><li>Ang, A. L., F&#246;lsch, H., Koivisto, U. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Journal of cell biology. 163 (2), 339-339 (2003).</li><li>Kroschewski, R., Hall, A., Mellman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature cell biology. 1 (1), 8-8 (1999).</li><li>Moskalenko, S., Henry, D. O., Rosse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature cell biology. 4 (1), 66-66 (2002).</li><li>Schuck, S., Gerl, M. J., Ang, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Traffic (Copenhagen, Denmark). 8 (1), 47-47 (2007).</li><li>Nokes, R. L., Fields, I. C., Collins, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Journal of cell biology. 182 (5), 845-845 (2008).</li><li>Collins, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cell.">Molecular cell.</a> 12, 1064-1064 (2003).</li><li>Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Science (New York, N.Y. 279 (5350), 509-509 (1998).</li><li>Scales, S. J., Pepperkok, R., Kreis, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell. 90 (6), 1137-1137 (1997).</li><li>Keller, P., Toomre, D., Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature cell biology. 3 (2), 140-140 (2001).</li></ol>

No conflicts of interest declared.

The most critical steps for a successful microinjection experiment are the quality and purity of the DNA and the polarity of your cells. Without polarized cells, your injection control will already have mistargeted VSVG and the experiment cannot be used. If the DNA is of poor quality, the DNA may clog the injection needle leading to poor or no expression of the desired protein at all. Also, it is advisable to use expression plasmids that are known to lead to high expression levels such as pRKV.
<p class="jove_content...

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Axiovert 200 Microscope with heated stage</td> 
			<td>Carl Zeiss Inc</td>
			<td>Custom order</td>
		</tr>
		<tr>
			<td>Injectman NI2 Femtojet micromanipulator</td>
			<td>Eppendorf</td>
			<td>Custom order</td>
		</tr>
		<tr>
			<td>Femtotips II (Microinjection needles)</td>
			<td>Eppendorf</td>
			<td>930000043</td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
		</tr>
		<tr>
			<td>Clear 12-mm transwell filter supports</td>
			<td>Corning Costar</td>
			<td>3460</td>
		</tr>
		<tr>
			<td>Endotoxin-free plasmid maxiprep kit</td>
			<td>Sigma-Aldrich</td>
			<td>NA0400</td>
		</tr>
	</tbody>
</table>

analyzing the function of small gtpases by microinjection of plasmids into polarized epithelial cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

Watch this Scientific Journal Video about Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells at JoVE.com

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

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11.3K 次观看.  Northwestern University. 本文详细介绍了在极化上皮细胞，利用显微注射技术的小GTP酶的过度表达和分析所涉及的程序。

视频: Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...