Name: intranuclear microinjection of dna into dissociated adult mammalian neurons
Uploaded: 2009-12-10T17:10:00
Duration: 13 min 39 s
Description: Watch this Scientific Journal Video about Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons at JoVE.com

Our laboratory research is supported by the Intramural Research Program of the NIH, National Institute on Alcohol Abuse and Alcoholism.

I. Preparation for injection
This section describes the preparation steps that need to be taken prior to injecting cDNA into the nucleus of neurons. The isolation procedure for neurons is beyond the scope of this article, but it is important to have neurons dissociated into individual cells and adhering well to the bottom surface of 35 mm cell culture dishes (see Discussion for helpful tips to achieve this). Although a variety of different neurons could potentially be used, peripheral ganglia such as the superior cervical ganglia (SCG) or the dorsal root ganglia are preferred for dissection because they are conveniently accessible and yield a large number of neurons. Additional advantages of using SCG neurons are that they are a relatively homogenous population of cells and well-characterized1,2,3,4. 
A. Preparation of plasmid cDNA for injection
<ol>
 <li>To prevent contamination or breakdown of DNA, gloves should be worn during the preparation. </li>
 <li>DNA encoding the protein of interest is subcloned into a suitable mammalian expression vector, such as pCI (Promega, Madison, WI), under the regulation of the highly and constitutively active cytomegalovirus (CMV) promoter. If the protein is not labeled, a reporter plasmid, encoding EGFP for example (pEGFP-N1, BD Biosciences Clontech, Palo Alto, CA), is co-injected to confirm successful injection and expression. DNA is isolated using a high quality separation column (e.g. QIAfilter Midi-prep kit, Qiagen, Chatsworth, CA) and further purified using a centrifugal filter unit with a PVDF filter (0.1 &mu;m, Millipore, Bedford, MA) to remove particulates in the plasmid preparation, which can obstruct injection pipets during the injection process. Plasmids are stored at -20&deg;C at a concentration of approximately 1 &mu;g/&mu;l in an appropriate DNA storage buffer (e.g. TE buffer: 10 mM Tris, 1 mM EDTA, pH 8.0). </li>
 <li>Immediately prior to microinjection, plasmid cDNAs are diluted and mixed to the desired final concentration in TE buffer or H2O. Typically, 5-10 ng/&mu;l of the reporter gene is sufficient to label injected cells, and the total concentration of cDNA to be injected should not exceed 200 ng/&mu;l since DNA is viscous at high concentrations and can clog injection pipets. A small volume of cDNA for injection (5-10 &mu;l) is prepared by mixing drops of solution on the clean surface of a small piece of Parafilm (side underneath the paper backing). Mix the drops of solution together by pipeting up and down several times with a pipetor and avoid introducing air bubbles during mixing.</li>
 <li>Using a microloader pipet tip (Eppendorf, Brinkmann Instruments, Westbury, NY), transfer the cDNA solution to a specially prepared hematocrit tube (Fisher Scientific, Pittsburgh, PA). See materials section for instructions on preparing hematocrit tubes (Materials Table). Place the hematocrit tube containing the cDNA injection solution into a 1.5 ml microcentrifuge tube. </li>
 <li>Centrifuge the tube for 15-30 min at 10 000 g in a microcentrifuge equipped with a fixed-angle or swinging bucket rotor (Eppendorf), at room temperature (20-24&deg;C) to sediment particles in the cDNA solution that may block the microinjection pipet. The cDNA injection solution can be kept at room temperature during the injection session. </li>
</ol>
B. Fabrication of injection pipets
<ol>
 <li>Disposable glass microinjection pipets are commercially available (e.g. Femtotips, Eppendorf), which are convenient but expensive ($10 per microinjection pipet). Injection pipets can be manufactured in the laboratory using a programmable P-97 Flaming Brown pipet puller (Sutter Instrument Company, Novato, CA) and filamented, thin-walled borosilicate glass capillary tubes (World Precision Instruments, Sarasota, FL). This option is more cost-effective and allows for control of microinjection pipet shape and size. </li>
 <li>A two-stage program is used to pull narrow microinjection pipet tips. Since the opening of microinjection pipets is too narrow to examine under a light microscope, the quality of injection pipets have to be evaluated directly by injecting a few cells before the program settings are finalized. The following are approximate settings for heat, pull, velocity and time settings: (pull 1) 600, 115, 15, 250; (pull 2) 640, 130, 65, 200. These settings vary with different batches of glass and by the condition of the heating filament in the puller, and should be adjusted as needed. For more details see5.</li>
 <li>Pull several (2-5) injection pipets per dish of neurons to be injected, in case of damage or problems during the injection session. Store injection pipets in a covered container to prevent dust from entering the microinjection pipet tip. </li>
</ol>
II. Intranuclear microinjection
This section details the process of injecting cDNA into the nucleus of neurons. A basic microinjection set-up includes an inverted phase-contrast microscope (e.g. Nikon Diaphot TMD, Nikon, Melville, NY) to visualize the process, micromanipulator (e.g. Eppendorf 5171) to control the movement of the injection pipet and a pressure injector (e.g. Eppendorf FemtoJet Express) to expel the cDNA solution from the pipet. Connecting the pressure injector to the micromanipulator allows for the synchronization of the latter two processes during injections. Other optional, but highly recommended, components include a mounted video monitoring system (CCD camera, Cohu Inc., San Diego, CA; black and white video monitor, Sony Corporation, Tokyo, Japan). This system is not an absolute requirement for injections; however, the black and white monitor offers high contrast for improved visualization of the cell nucleus and offers a more comfortable viewing experience during long injection sessions. In addition, a laptop computer running software to operate a hand-held controller can be utilized to semi-automate the injection process (see Discussion for details).
The ideal time to inject SCG neurons is 3-6 hours post-dissociation. At this stage, the cells are spherical, tightly attached to the bottom of the dish, and the nucleus is clearly visible, under a phase-contrast microscope, as a central round organelle with a dark membrane, containing a single or multiple nucleoli (Figure 1A).
<ol>
 <li>Place a dish of dissociated neurons on the center of the microscope stage. Adjust the focus and optimize the phase contrast optics of the microscope so the nuclei of neurons are clearly visible. </li>
 <li>Pipet 2 &mu;l of prepared cDNA solution into a microinjection pipet using a microloader pipet tip. Avoid drawing up solution near the bottom of the hematocrit tube since this may stir up particulates that settled during centrifugation. The filament in the microinjection pipets should aid in drawing solution to the tip, but if small air bubbles persist, gently flick the side of the glass to dislodge them. </li>
 <li>Insert the microinjection pipet into the capillary holder of the pressure injector and then secure the holder to the micromanipulator. The holder is fixed at a 45&deg; angle. </li>
 <li>Lower the microinjection pipet into the dish. As the pipet enters the culture medium, a meniscus is formed that affects the refraction of light and lowers the image quality of the neurons. To alleviate this problem, the phase ring turret can be slightly offset until the nuclei of neurons are clearly visible again. </li>
 <li>Some pressure injection systems have a "clean" function that applies maximum air pressure from the microinjection pipet tip for a short duration. Use this function to clear the pipet of debris before beginning and during injections if the tip appears clogged. If dispelled fluid is not visible on the screen while using this function, replace with a new injection pipet. </li>
 <li>Center the microinjection pipet in the viewing monitor and align the tip beside a neuron and in the same focal plane as the nucleolus. Set this position as the lower z-axis limit on the micromanipulator. This position is the depth the microinjection pipet will advance to during injections. Now position the tip approximately 30 &mu;m above this point so the microinjection pipet clears the top of neurons. </li>
 <li>The "inject" mode of an Eppendorf 5171 micromanipulator can be defined with the following settings to perform intranuclear injections. The inject function is set on, whilst the impale function is left off. A diagonal injection path (along the x- and z-axis) produces the least amount of cell damage, and thus the axial mode should be used for injections. An injection speed of 300 &mu;m/s is sufficient, and synchronize pressure build-up when the microinjection pipet reaches the set z-axis limit. </li>
 <li>The pressure injector controls the quantity of cDNA solution injected into the nucleus. In the "automatic" injection mode, the delivery of DNA is time-controlled and initiated by the connected micromanipulator. The injection pressure (Pi) should be set between 100 to 200 hectopascals (hPa; 1 hPa ~ 0.015 psi); higher injection pressures do not improve success rates or quality of injections and may indicate other problems with the microinjection pipet tip size or DNA purity. An injection duration (ti) of 0.3 s and a compensation pressure (Pc) of 30 hPa, which supplies a constant positive pressure to avoid entry of occluding particles from the culture medium into the pipet tip, should be used. </li>
 <li>Position the neuron to be injected under the tip of the microinjection pipet using the microscope&#x2019;s xy-axis stage control. Align the tip of the pipet above the center of the nucleus by focusing back and forth between the tip and the nucleoli. </li>
 <li>Inject the nucleus by pressing the inject button (on the micromanipulator). For the injection step, the movement of the microinjection pipet and the release of cDNA solution are controlled by the micromanipulator. It is difficult to confirm successful intranuclear injections by eye, but the injection of the relatively low viscosity cDNA solution (compared to the viscous nuclear environment), often appears as a white plume under phase-contrast optics and can be used as an indicator of injection into the nucleus. Sometimes the microinjection pipet does not penetrate the nuclear membrane and simply nudges the nucleus. A second injection attempt at the same position may lead to a successful injection of DNA into the nucleus. Swelling of the cell is usually indicative of unintentional cytoplasmic injection. Also, significant nuclear swelling is not well tolerated by neurons and usually results in cell death soon afterwards; thus, the injection pressure and duration should not exceed the suggested values. </li>
<li>Continue injecting neurons by repositioning the microscope stage to align the next neuron under the microinjection pipet. Systematically navigate through the dish to inject approximately 50-100 nuclei before returning the dish of neurons to the incubator for overnight incubation. Successfully injected neurons can be identified the following day by expression of the reporter gene.</li>
</ol>
III. Representative Results
<img src="/files/ftp_upload/1614/1614fig1.jpg" alt="Figure 1" /> Figure 1: Superior cervical ganglion (SCG) neurons. Phase contrast image of SCG neurons 3 hours post-dissociation (A). At this stage, the nucleus is clearly visible which aids alignment of the microinjection pipet tip. The nucleus appears in the center of neurons with a single (left) or multiple (right) dark nucleoli. SCG neurons 16 hours following injection of EGFP cDNA into the nucleus (B). Left, SCG neurons viewed under phase-contrast illumination. Right, epifluorescent image of the same neurons showing successful injection and resulting EGFP expression in one neuron. White scale bar is 20 &mu;m.
<img src="/files/ftp_upload/1614/1614figure2tmb.jpg" alt="Figure 2" border="0" /> Figure 2: Whole-cell recordings of currents through heterologously-expressed G-protein coupled inwardly rectifying K+ (GIRK) channels from SCG neurons. GIRK currents (IGIRK) were evoked by a 200 ms voltage ramp from -140 to +40 mV from a holding potential of -60 mV (inset of A) following activation of endogenous &alpha;2-adrenoceptors by norepinephrine (NE, 10 &mu;M). Superimposed traces are recordings from the same neuron and indicate before (black) and after application of either NE alone (blue) or NE plus 10 mM Ba2+ (red). Dashed lines indicate the zero current level. For recording IGIRK, the external solution contained (in mM) 130 NaCl, 5.4 KCl, 10 HEPES, 10 CaCl2, 0.8 MgCl2, 15 glucose, 15 sucrose, and 0.0003 TTX, adjusted to pH 7.4 with NaOH. The internal recording solution contained (in mM) 135 KCl, 11 EGTA, 1 CaCl2, 2 MgCl2, 10 HEPES, 4 MgATP, and 0.3 Na2GTP, adjusted to pH 7.2 with KOH. The lack of current elicited by the voltage ramp in the presence of NE in uninjected SCG neurons (A) demonstrates that SCG neurons do not express endogenous GIRK channels. Successful injection of plasmid cDNA encoding a functional GIRK channel11 gives rise to large currents during the voltage ramp when exposed to NE (B, left). Currents have the typical characteristics of currents through GIRK channels: activation by a G-protein coupled receptor agonist (NE), inward rectification, and block of currents by the putative GIRK channel blocker, Ba2+ (B, right red trace). Open and filled circles represent IGIRK at the holding and peak current, respectively. Please <a href="https://www.jove.com/files/ftp_upload/1614/1614figure2.jpg"> click here</a> to see a larger version of figure 2.

<ol>
	<li>Eccles, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+action+potential+of+the+superior+cervical+ganglion.">The action potential of the superior cervical ganglion.</a> J Physiol. 85, 179-206 (1935).</li><li>Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-dependent+modulation+of+N-type+calcium+channels+by+G-protein+&#x03B2;&#x03B3;+subunits.">Voltage-dependent modulation of N-type calcium channels by G-protein &#x03B2;&#x03B3; subunits.</a> Nature. 380, 255-258 (1996).</li><li>Schofield, G. G., Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+currents+of+acutely+isolated+adult+rat+superior+cervical+ganglion+neurons.">Potassium currents of acutely isolated adult rat superior cervical ganglion neurons.</a> Brain Res. 485, 205-214 (1989).</li><li>Schofield, G. G., Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+and+calcium+currents+of+acutely+isolated+adult+rat+superior+cervical+ganglion+neurons.">Sodium and calcium currents of acutely isolated adult rat superior cervical ganglion neurons.</a> Pflugers Arch. 411, 481-490 (1988).</li><li>Dean, D. A., Goldman, R. D., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+4.">Chapter 4.</a> Live Cell Imaging: a Laboratory Manual. 1, 51-66 (2005).</li><li>Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterologous+expression+of+receptors+and+signaling+proteins+in+adult+mammalian+sympathetic+neurons+by+microinjection.">Heterologous expression of receptors and signaling proteins in adult mammalian sympathetic neurons by microinjection.</a> Methods Mol Biol. 83, 191-202 (1997).</li><li>Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+G-protein+signaling+components+in+adult+mammalian+neurons+by+microinjection.">Expression of G-protein signaling components in adult mammalian neurons by microinjection.</a> Methods Mol Biol. 259, 167-181 (2004).</li><li>Ikeda, S. R., Jeong, S. W. <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+RGS-insensitive+G&#x03B1;+subunits+to+study+endogenous+RGS+protein+action+on+G-protein+modulation+of+N-type+calcium+channels+in+sympathetic+neurons..">Use of RGS-insensitive G&#x03B1; subunits to study endogenous RGS protein action on G-protein modulation of N-type calcium channels in sympathetic neurons..</a> Methods Enzymol. 389, 170-189 (2004).</li><li>Washbourne, P., McAllister, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+gene+transfer+into+neurons.">Techniques for gene transfer into neurons.</a> Curr Opin Neurobiol. 12, 566-573 (2002).</li><li>Zhang, Y., Yu, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+microinjection+technology+in+cell+biology.">Single-cell microinjection technology in cell biology.</a> Bioessays. 30, 606-610 (2008).</li><li>Vivaudou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+G-protein+regulation+of+GIRK1+and+GIRK4,+the+two+subunits+of+the+KACh+channel,+using+functional+homomeric+mutants.">Probing the G-protein regulation of GIRK1 and GIRK4, the two subunits of the KACh channel, using functional homomeric mutants.</a> J Biol Chem. 272, 31553-31560 (1997).</li></ol>

Common problems encountered and suggestions:
As mentioned earlier, successful nuclear injections depend on having cells adherent to tissue culture plates. Postponing the start of injections a few hours following the cell dissociation procedure allows neurons to adhere to the bottom of dishes. In addition, coating dishes with 0.1 mg/ml of high molecular weight poly-L-lysine (Sigma, St. Louis, MO) aids adhesion of neurons to the bottom surface of dishes.
Blockage of microinjection pipets, especially when attempting to inject a high concentration of DNA, can be a frequent problem. Using high quality separation columns to isolate and purify plasmid DNA, and performing the additional purification steps mentioned (spin filters, spinning in hematocrit tubes) can help to remove particulates prior to injecting. During injections, the &#x201C;clean&#x201D; function of the pressure injector can be used (for 0.2 s is sufficient); but if that does not resolve the problem, a new injection pipet should be used. If the majority of microinjection pipets become clogged during an injection session, consider altering the settings of the glass pipet puller to produce microinjection pipet tips with a larger opening.
Another issue that arises during nuclear injections is the unevenness of the bottom surface of tissue culture dishes. This variability directly affects the targeting accuracy of injection pipet tips to the nucleus since the depth the pipet traverses during injections is fixed. Therefore, this z-axis limit must be adjusted during the course of injections within the same dish. It will be apparent when an adjustment is necessary: there will be no indication of nuclear injection (no white plume in the nucleus or the cell is missed entirely), or the injection pipet tip comes close to the bottom of the dish (compressing the cell or even crashing the pipet tip into the bottom of the dish). Glass-cloning cylinders (O.D. 10 mm, Cat. No. 2090-01010, Bellco Glass, Vineland, NJ) can be used during the plating of neurons to restrict them in a smaller, more confined area; therefore minimizing the distance required to move the injection pipet between injections. These cloning cylinders are removed prior to performing microinjections.
Drawbacks of technique:
Intranuclear microinjections are technically demanding, which require patience and a high degree of manual dexterity by the operator. Proficiency with this technique, like with any other skill, comes with practice. Thus, it is advised to practice nuclear injections of the reporter gene alone before attempting actual experiments. To further assist the injection process, it may be possible to consolidate the steps of the micromanipulator and pressure injector into a computer program. Programs such as Igor Pro (WaveMetrics, Lake Oswego, OR) can be utilized to store microinjection settings and to program multifunctional controllers (ShuttlePro, Contour Design, Windham, NH) to semi-automate the injection process (see 6,7,8 for details). 
 Another drawback of the intranuclear microinjection technique is the low success rate. Approximately 10-20% of attempted nuclear injections lead to protein expression. Whilst this efficiency may be sufficient for applications that do not require a large number of expressing cells, electrophysiology for example, for various biochemical assays this may not be sufficient. 
Applications of technique:
Despite its drawbacks, intranuclear microinjection of DNA is an extremely useful technique for heterologously expressing proteins in neurons.
High levels of protein expression are achieved with nuclear microinjections because of the large number of plasmids released into the nucleus during injections, a highly active CMV promoter regulating gene transcription, and long stability of plasmids in the nucleus. Protein over-expression is especially useful in dominant-negative experiments where high levels of heterologous proteins are required to overwhelm the endogenous protein. Another advantage of intranuclear injections is the ability to deliver a consistent proportion of cDNA constructs to the nucleus when multiple constructs are injected. With other transfection methods, it is difficult to reproducibly introduce the same proportion of each DNA construct into different cells within the same dish and between transfections. Direct injection of cDNA solution into the nucleus eliminates this source of variability and allows the ratio of expressed proteins to be titered effectively.
Traditional transfection methods have limited effectiveness in post-mitotic cells because of low incorporation of DNA into the nucleus9 and chemical toxicity associated with transfection reagents10. Nuclear microinjections overcome these issues by introducing genetic material mechanically into the nucleus. Although this technique is more involved, the ability to study the effect of a protein in a functional biological system, complete with endogenous signaling pathways, more than compensates for the laborious nature of this technique.
All experimental procedures on animals were performed in accordance with the National Institutes of Health&#x2019;s Guidelines for Animal Care and Use.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ultrafree-MC Filter Unit</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC30VV00</td>
<td>Durapore PVDF filter; 0.1 &mu;m pore-size</td>
</tr>
<tr>
<td>TE buffer</td>
<td>&nbsp;</td>
<td>Quality Biological, Inc</td>
<td>351-010-131</td>
<td>10 mM Tris, 1 mM EDTA, pH 8.0</td>
</tr>
<tr>
<td>Micro-hematocrit capillary tubes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>22-362-574</td>
<td>Unheparinized Preparation: 
Soak in 100% ethanol overnight. Wash thoroughly with deionized water three times and dry bake glass in an oven (95&deg;C). Cut the cleaned hematocrit tubes into 3-4 cm length pieces using a diamond-tipped scriber, and seal one end closed with a flame and forceps to hold the glass. Store in a clean glass beaker and cover to prevent dust build-up. </td>
</tr>
<tr>
<td>Microcentrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5417 R</td>
<td>With a fixed angle or swinging-bucket rotor</td>
</tr>
<tr>
<td>Microloader 20 &mu;l pipet tips</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5242 956.003</td>
<td>&nbsp;</td>
<tr>
<td>Microinjection capillary glass</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TW120F-4</td>
<td>Thin-walled with filament, 1.2 mm outer-diameter, 0.9 mm inner-diameter, 100 mm length</td>
</tr>
<tr>
<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument Co.</td>
<td>P-97</td>
<td>With platinum-iridium box filament (part # FB330B)</td>
</tr>
<tr>
<td>Nikon Diaphot TMD Inverted Microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>20x and 40x phase-contrast objective lenses, mounted on a vibration isolation table</td>
</tr>
<tr>
<td>Charge-coupled device (CCD) camera </td>
<td>&nbsp;</td>
<td>Cohu Inc.</td>
<td>6410</td>
<td>&nbsp;</td>
<tr>
<td>Video monitor</td>
<td>&nbsp;</td>
<td>Sony Corporation</td>
<td>PVM-122</td>
<td>Black and White, 9&#x201D; screen </td>
</tr>
<tr>
<td>Micromanipulator system</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5171</td>
<td>&nbsp;</td>
<tr>
<td>Microinjection Unit</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>FemtoJet Express</td>
<td>&nbsp;</td>
<tr>
<td>Microinjection controller</td>
<td>&nbsp;</td>
<td>Contour Design</td>
<td>S-PROV2</td>
<td>Programmable multimedia controller</td>
</tr>
<tr>
<td>Igor Pro</td>
<td>&nbsp;</td>
<td>WaveMetrics</td>
<td>Version 4.05A</td>
<td>Command program to control the microinjection controller written by S. Ikeda</td>
</tr>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons at JoVE.com

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

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 ...

intranuclear-microinjection-dna-into-dissociated-adult-mammalian

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Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

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 ...

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.

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 ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...