The author would like to acknowledge the following people. The staff of the University of Maryland Insect Transformation Facility: Channa Aluvihare, Robert Alford, and Daniel Gay. Without their dedicated work, the Insect Transformation Facility would not exist. Vanessa Meldener-Harrell for proofreading this manuscript.

NOTE: The protocol as described below uses the Sutter BV-10 microcapillary beveled. However, this protocol can be modified for use with any model microcapillary beveled.
1. Assembly of regulator, pressure gauge, and air supply tubing
<ol>
	<li>Cut a section of urethane tubing for the connection from the air supply to the base of the regulator (R; Section 1, Figure 1). The length of this section will depend on the distance from the air supply to the regulator, which will be located next to the beveler.</li>
	<li>Slide a hose clamp onto the urethane tubing, then push a hose connector into the end of the tubing. Make sure the hose connector is fully inserted by placing the hose connector against a hard surface. While holding the tubing close to the hose connector, press the tubing firmly into the hose until the hose connector is fully inserted. Together, the hose clamp and hose connector form the connection HC (Figure 1).</li>
	<li>Slide the hose clamp up to the end of the tubing where the hose connector is inserted. Rotate the hose clamp over the inserted barb end so that it forms an airtight connection.</li>
	<li>Screw the hose connector into the base of the regulator (R) by hand, then finish tightening the connection with a small wrench. Make sure the connection is airtight but not overtight.</li>
	<li>In the side port of the regulator, screw in a T connector (T) by hand, then use a small wrench to finish tightening.</li>
	<li>Cut a small piece of urethane tubing (Figure 1, Section 2), approximately 3-5 cm in length. Place two hose clamps onto the piece of tubing, then insert a hose connector into each end of the tubing and finish the connections (HC) as described in steps 1.2-1.4 for each end of the tube.</li>
	<li>Screw one end of the short tube into the base of the pressure gauge, then finish tightening with a wrench as in step 1.5.</li>
	<li>Screw the other end of the short tube into one of the ports on the T connector (T) connected to the regulator in step 1.5.</li>
	<li>Cut a section of urethane tubing (Figure 1, Section 3) for the connection between the regulator and needle holder (NH). The size of this section will depend on the distance from the beveler to the location of the regulator.</li>
	<li>Place a hose clamp onto the tubing section, then insert a hose connector as described in steps 1.2-1.4.</li>
	<li>On the other side of this section of tubing, place the female luer connector (LC) that is supplied with the needle holder (NH).</li>
	<li>Remove the retaining clamp and the nylon washer (Figure 2, f) from the manipulator. The retaining clamp and washer are designed to hold a glass microcapillary only, they are not designed to hold the additional weight of a microcapillary holder, threaded rod, and air supply tubing.</li>
	<li>Cut a rectangular piece of rubber packing sheet 1.5 cm x 2 cm to wrap the threaded rod in the bicycle fender clamp. This will help hold the threaded rod and needle holder more securely in the bicycle fender clamp. Using two needle nose pliers, open the bicycle fender clip, fold the rectangular piece of rubber packing sheet over the threaded rod 3.5 cm from one end of the rod so that it forms a U over the rod. Insert the rubber sheet-covered threaded rod into the opened bicycle fender clamp and use the pliers to close the clamp around the rod and rubber sheet. Install the bicycle clamp and threaded rod assembly onto the manipulator bolt, replace the retaining clamp without the nylon washer, and tighten the retaining clamp until it securely holds the threaded rod assembly.</li>
	<li>Thread the needle holder onto the threaded rod. Make sure that the luer connector ends in a position that will not bind the urethane tubing when it is connected (Figure 2, g).</li>
	<li>Connect the female luer connector to the male luer connector (Figure 2, g) of the needle holder (Figure 2, d).</li>
	<li>Connect the free end of Figure 1, Section 1 tube to the air supply (this connection will vary based on the air supply used). The air supply should be clean, dry air, and free of oil residue. The air supply can be from a house air source or gas cylinder, either compressed air or Nitrogen.</li>
</ol>
2. Beveling borosilicate needles
<ol>
	<li>Pull microinjection needles using borosilicate glass micro capillaries with the following settings on the instrument: Heat: 305, Fil: 4, Vel: 70, Del: 235, Pul: 160, with Loop time of 12.24.</li>
	<li>Assemble the grinding assembly according to the manufacturer&#39;s instructions, consisting of the abrasive plate and the retaining ring with magnets12. 
	NOTE: The abrasive plate may need to be wrapped with a thin strip of transparent film to prevent premature leakage of Photo-Flo (wetting agent) from the abrasive plate. This is only needed if the wetting agent solution prematurely leaks down into the pedestal oil, causing the grinding plate to stop rotating. The wetting agent reduces the risk of drying marks on the glass needles after beveling. The use of a wetting agent is not critical to the beveling process, and water may be substituted.</li>
	<li>Place 10 drops of pedestal oil onto the pedestal&#39;s optically flat surface and place the grinding assembly on top. Start the grinding assembly.</li>
	<li>Turn on the light source to illuminate the surface. Ensure the light source is positioned behind the beveled and shines at an angle of 45&#176; to the abrasive plate and needle. The illumination angle is necessary so that a shadow of the needle is easily seen. At 90x magnification, rotate the microscope head in place. Briefly stop the abrasive plate spinning and focus the microscope on the surface of the abrasive plate.</li>
	<li>Add 1% wetting agent to the wick until the wick is completely wet. Add 1% wetting agent to the surface of the abrasive plate. Ensure the wetting agent covers the abrasive surface but does not leak on to the black retaining ring.</li>
	<li>Place the pre-wet wick onto the surface of the abrasive plate as it is spinning. Ensure the wick is on the left side of the abrasive plate (as you look down from the top) and stretches from 11 o&#39;clock to 6 o&#39;clock (with the plate as a clock face). Ensure the wick does not ride on the black portion of the retaining ring.</li>
	<li>Insert a needle into the needle holder (Figure 2, d) and tighten the retaining ring to hold the needle in place. Open the regulator (Figure 1, R) and increase the pressure to 24 psi.</li>
	<li>Raise the needle holder by rotating the course adjustment knob (Figure 2, a) Make sure the needle is raised high enough that it is higher than the surface of the rotating abrasive plate, then rotate the entire manipulator so that the needle swings into place above the rotating abrasive plate. The needle to be beveled should be placed onto the rotating abrasive plate oriented such that the rotation of the plate moves away from the tip of the needle (Figure 3A)</li>
	<li>Watching from the side, use the coarse adjustment knob (Figure 2, a) to lower the needle toward the abrasive plate surface. Stop when the needle is almost touching the surface of the liquid.</li>
	<li>Use the zoom to lower the magnification of the microscope, then move the microscope so that the needle is in the center of the field of view. Once in the center of view, increase the magnification, adjusting the manipulator&#39;s position so that the needle tip stays in the center of view. Once at maximum magnification, stop the grinding plate and focus the microscope on the surface of the abrasive plate, then immediately restart the rotation of the plate once the surface is in focus. The needle may not be in view at this point.</li>
	<li>Using the manipulator coarse adjustment knob, lower the needle toward the abrasive plate. In the field of view, an image of the needle and a shadow(s) of the needle will be visible. When the needle and the shadow(s) of the needle are close to touching, switch to the manipulator fine adjustment knob and continue to lower the needle until the needle and its shadow(s) appear to touch. At this point, read the caliper (Figure 2, c) and note the reading. The surface of the abrasive plate is at or below this caliper reading. 
	NOTE: It is difficult to see when the needle touches the surface of the rotating abrasive plate, so the needle may not actually be touching the abrasive plate at this point.</li>
	<li>Allow the needle to stay at this level of caliper reading for 5-10 s.</li>
	<li>Using the manipulator fine adjustment knob, raise the needle, making sure it stays underneath the surface of the wetting agent. Stop the rotation of the abrasive plate for a few seconds and observe whether bubbles escape from the needle tip. If bubbles are present, proceed to step 2.15. If bubbles are not present, proceed to step 2.14.</li>
	<li>Move the needle back to the caliper reading using the manipulator fine adjustment knob, then move it a little lower and take a new caliper reading, then repeat steps 2.12-2.13.</li>
	<li>Start the abrasive plate again to see if bubble formation is observable while the plate is rotating. If evidence of bubble formation is visible during plate rotation, the opening of the needle tip is likely too large for sensitive embryo microinjections, such as mosquito embryo injections. If bubble formation is not visible during abrasive plate rotation, then the needle opening is ideal for use in procedures requiring a sharp and small opening needle.</li>
	<li>Use the manipulator course adjustment knob to raise the needle above the abrasive plate to a position that is high enough above the plate that the needle will not hit anything as the entire manipulator is rotated to move the needle away from the abrasive plate and microscope.</li>
	<li>Once the needle is in a position where it can be removed without hitting anything, lower the air pressure to zero, remove the needle, and place it in a needle storage box (a Petri dish with either double stick tape or modeling clay to hold the beveled needles) until use.</li>
</ol>
3. Determining the relative opening size of the beveled needle
NOTE: Determining the beveled needle&#39;s relative opening sizes is done in step 2.13. The steps below describe this process further.
<ol>
	<li>Once bubbles are observed in step 2.13, with the abrasive plate not rotating, slowly decrease the air pressure by rotating the regulator adjustment knob (Figure 1, R) until bubbles stop flowing from the tip of the needle. Note the pressure at which bubbles stop flowing from the tip.</li>
	<li>Increase the air pressure until bubbles are flowing again from the needle tip. The higher the pressure, the smaller the opening of the needle.</li>
	<li>Proceed with moving the needle to a position where it can be safely removed steps 2.16-2.17.</li>
</ol>

Protocol for Wet Beveling of Microinjection Needles Under Constant Air Pressure

<ol>
	<li>Rubin, G. M., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+Drosophila+with+transposable+Element+Vectors.">Genetic transformation of Drosophila with transposable Element Vectors.</a> Science. 218 (4570), 348-353 (1982).</li><li>O'Brochta, D. A., Atkinson, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+systems+in+insects.">Transformation systems in insects.</a> Methods Mol Biol. 260, 227-254 (2004).</li><li>Handler, A. M., James, A. 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> Insect Transgenesis: Methods and Applications. , (2000).</li><li>Harrell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+embryo+microinjection.">Mosquito embryo microinjection.</a> Cold Spring Harbor Protocols. , (2023).</li><li>Allen, M. L., O'Brochta, D. A., Atkinson, P. W., Levesque, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable,+germ-line+transformation+of+Culex+Quinquefasciatus+(Diptera:+Culicidae).">Stable, germ-line transformation of Culex Quinquefasciatus (Diptera: Culicidae).</a> J Med Entomol. 38 (5), 701-710 (2001).</li><li>Grossman, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+transformation+of+the+malaria+vector,+Anopheles+gambiae,+with+the+piggyBac+transposable+element.">Germline transformation of the malaria vector, Anopheles gambiae, with the piggyBac transposable element.</a> Insect Mol Biol. 10 (6), 597-604 (2001).</li><li>Adelman, Z. N., Jasinskiene, N., James, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+applications+of+transgenesis+in+the+yellow+fever+mosquito,+Aedes+aegypti.">Development and applications of transgenesis in the yellow fever mosquito, Aedes aegypti.</a> Mol Biochem Parasitol. 121 (1), 1-10 (2002).</li><li>Perera, O. P., Harrell, R. A., Handler, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-line+transformation+of+the+South+American+malaria+vector,+Anopheles+albimanus,+with+a+piggyBac/EGFP+transposon+vector+is+routine+and+highly+efficient.">Germ-line transformation of the South American malaria vector, Anopheles albimanus, with a piggyBac/EGFP transposon vector is routine and highly efficient.</a> Insect Mol Biol. 11 (4), 291-297 (2002).</li><li>Harrell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+embryo+microinjection+under+halocarbon+oil+or+in+aqueous+solution.">Mosquito embryo microinjection under halocarbon oil or in aqueous solution.</a> Cold Spring Harb Protoc. , (2023).</li><li>Louradour, I., Ghosh, K., Inbar, E., Sacks, 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=CRISPR/Cas9+mutagenesis+in+Phlebotomus+papatasi:+The+immune+deficiency+pathway+impacts+vector+competence+for+Leishmania+major.">CRISPR/Cas9 mutagenesis in Phlebotomus papatasi: The immune deficiency pathway impacts vector competence for Leishmania major.</a> mBio. 10 (4), e01941 (2019).</li><li>Tamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+transformation+of+the+silkworm+Bombyx+mori+L.+using+a+piggyBac+transposon-derived+vector.">Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.</a> Nat Biotechnol. 18 (1), 81-84 (2000).</li><li>. BV-10 Micropipette Beveler Operation Manual Rev. 3.00 Available from: <a target="_blank" href="https://www.manualslib.com/manual/2073788/Sutter-Instrument-Bv-10.html">https://www.manualslib.com/manual/2073788/Sutter-Instrument-Bv-10.html</a> (2018)</li></ol>

The author has nothing to disclose.

Genetic modification of mosquitoes relies on precise microinjection of the modification materials (plasmids, guide RNAs, or proteins) into pre-blastoderm embryos3,4,5,6,7,8. Crucial to this process are sharp needles that easily pierce the embryo during injection2,4. A...

Insect genetic modification is a process originally developed in Drosophila by Rubin and Spradling, and over the years, this process has been modified to create genetic modifications in other species1. The process relies on the precise delivery of modification components microinjected into embryos at a specific window of time and location within the developing embryo2,3,4. Sharp microinjection needles are a critical tool in the process of genetic modification of some insects, such as mosquitoes4,5,6,7,8,9 and sand flies10 while not as critical for other insects, such as silkworms11. Sharp needles are often a key factor between success and failure when trying to create a genetically modified insect2,4. Typically, microcapillary glass needles are pulled by heating glass to the point where the glass becomes elastic, allowing the capillary to be pulled into a tapering closed tip needle. Before the needle can be used, it needs to be opened in a manner that creates a sharp tip for injection. Traditionally, needles are opened by brushing the needle tip gently against something (the edge of a slide/coverslip, or the embryo, etc.) that causes a small amount of glass to break off from the tip, randomly creating a sharp tip2,3. A slightly less random process is dry beveling, where the needle is quickly lowered onto an optically flat, spinning abrasive plate for a short period of time, causing a small amount of glass to be abraded from the needle tip, creating a sharp tip. Dry beveling is a little less random than brushing the needle tip against something. The protocol described below takes the beveling process a step further by supplying compressed air to the needle being beveled and beveling it under a liquid layer so that bubbles are visible as soon as the needle has been opened. This protocol details a method for producing reliably sharp microinjection needles. Beveling under a liquid layer is an improvement over randomly breaking the needle as described above, and dry beveling because the user receives feedback on the beveling process, allowing the person beveling to know when the needle is open and relatively how large the needle opening is. Knowing the relative opening size of the needle tip can allow the person beveling to create needles with different opening sizes. Various needle opening sizes have different advantages; for instance, larger needle opening sizes can accommodate injection mixes of high viscosity, while smaller opening needles cause less damage to the embryo being injected.
Air is supplied to the needle using a regulator and a system of urethane tubing based on a modified air-pressure regulated injection system2. While air is supplied to the needle at a constant pressure, the needle is beveled under a layer of liquid. The beveling process is comprised of a repeated five-step process: 1) lowering the needle to the abrasive surface, 2) allowing the needle to bevel for a short period of time, 3) raising the needle away from the abrasive plate while keeping it beneath the surface of the liquid layer, 4) stopping the spin of the abrasive plate to check for the appearance of bubbles. If no bubbles are present, then steps 1 through 4 are repeated until bubbles appear; 5) Once bubbles are present, the air pressure can be adjusted to determine the relative opening size of the needle. The lower the pressure needed to stop bubble formation, the larger the opening at the needle tip.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.0 mm O.D. microcapillaries</td>
			<td>World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beveler pedestal oil</td>
			<td>Sutter Instruments</td>
			<td>008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bicycle fender clip</td>
			<td>VeloOrange</td>
			<td>R-clip 4-pack</td>
			<td>https://velo-orange.com/products/vo-r-clip-4-pack</td>
		</tr>
		<tr>
			<td>Boom Stand Microscope</td>
			<td>AmScope</td>
			<td></td>
			<td>AMScope 3.5X-90X Trinocular LED Boom Stand Stereo Microscope or equivalent</td>
		</tr>
		<tr>
			<td>BV-10 Beveler</td>
			<td>Sutter Instruments</td>
			<td>BV-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond abrasive plate&#160;</td>
			<td>Sutter Instruments</td>
			<td>104F</td>
			<td>Diamond abrasive plate - extra fine (0.2 &#181; to 1.0 &#181; tip sizes)</td>
		</tr>
		<tr>
			<td>Gasket, Buna-N</td>
			<td>Clippard Instrument Laboratory, Inc.</td>
			<td>11761-2-pkg</td>
			<td>Used to seal connection on T&#160; or L connectors, if not already included with these pieces</td>
		</tr>
		<tr>
			<td>Hose Clamp</td>
			<td>Clippard Instrument Laboratory, Inc.</td>
			<td>5000-2-pkg</td>
			<td></td>
		</tr>
		<tr>
			<td>Hose connector</td>
			<td>Clippard Instrument Laboratory, Inc.</td>
			<td>CT4-pkg</td>
			<td>Need 5 hose connectors</td>
		</tr>
		<tr>
			<td>Microinjection Needle Holder</td>
			<td>World Precision Instruments</td>
			<td>MPH3-10</td>
			<td>Needle holder for 1mm outer diameter microcapillaries</td>
		</tr>
		<tr>
			<td>P-2000</td>
			<td>Sutter Instruments</td>
			<td></td>
			<td>Any needle puller</td>
		</tr>
		<tr>
			<td>Photo-Flo 200 Solution</td>
			<td>B&#38;H Photo, Video and Audio</td>
			<td>BH #KOPF200P&#160; MFR #1464510</td>
			<td>wetting agent</td>
		</tr>
		<tr>
			<td>Pressure Gauge</td>
			<td>Clippard Instrument Laboratory, Inc.</td>
			<td>PG-100</td>
			<td>0-100 psi gauge</td>
		</tr>
		<tr>
			<td>Reference wick</td>
			<td>Sutter Instruments</td>
			<td>X050300</td>
			<td></td>
		</tr>
		<tr>
			<td>Reference wick holder</td>
			<td>Sutter Instruments</td>
			<td>M100019</td>
			<td></td>
		</tr>
		<tr>
			<td>Regulator</td>
			<td>Clippard Instrument Laboratory, Inc.</td>
			<td>01-Mar</td>
			<td>Need #10-32 ports for connections</td>
		</tr>
		<tr>
			<td>Rubber Packing Sheet 6 inx 6 in</td>
			<td>Danco</td>
			<td>Model # 59849</td>
			<td></td>
		</tr>
		<tr>
			<td>T fitting</td>
			<td>Clippard Instrument Laboratory, Inc.</td>
			<td>15002-2-pkg</td>
			<td></td>
		</tr>
		<tr>
			<td>Threaded Bar</td>
			<td></td>
			<td></td>
			<td>Either a threaded rod or bar with threaded end. Threads must be 10-32.</td>
		</tr>
		<tr>
			<td>Urethane tubing</td>
			<td>Clippard Instrument Laboratory, Inc.</td>
			<td>URH1-0804-BLT-050</td>
			<td></td>
		</tr>
	</tbody>
</table>

wet beveling of microinjection needles utilizing constant air pressure for feedback on needle opening

Microinjection needles are a critical tool in the delivery of genome modification reagents, CRISPR components (guide RNAs, Cas9 protein, and donor template), and transposon system components (plasmids and transposase mRNA) into developing insect embryos. Sharp microinjection needles are particularly important during the delivery of these modifying agents since they help minimize damage to the embryo being injected, thereby increasing the survival of these embryos as compared to injection with non-beveled needles. Further, the beveling of needles produces needles that are more consistent from needle to needle as compared to needles opened by randomly breaking the needle tip by brushing the tip against an object (side of a coverslip, the surface of the embryo to be injected, etc.). The process of wet beveling of microinjection needles with constant pressure air delivered to the needle allows the person beveling the needle to know when the needle is open (presence of bubbles) and also gives a relative indication of how large a needle opening has been created. The relative opening size in the needle can be determined by adjusting the air pressure delivered to the needle until an equilibrium is reached and bubbles stop flowing from the tip of the needle. The lower the pressure at which the equilibrium is reached, the larger the needle size; and conversely, the higher the pressure, the smaller the needle size.

The procedure described above produces consistently sharp microinjection needles. Sharp needles are characterized by being able to insert into soft chorion insect embryos, such as mosquito embryos, with little to no resistance from the embryo membrane. When mosquito embryos are microinjected for genetic modification, the embryo membrane is relatively elastic. Pushing a dull needle against the embryo membrane will cause it to indent (Figure 4B). When the needle is pulled back, the membrane re...

Author Spotlight: Enhancing Microinjection Needle Quality by Wet Beveling

This protocol describes the assembly of a pneumatic system for the delivery of pressurized air to a needle during the process of needle beveling. The protocol further describes the beveling process for creating sharp microinjection needles and how to gauge the relative opening size of the needle.

Wet Beveling of Microinjection Needles Utilizing Constant Air Pressure for Feedback on Needle Opening

Microinjection needles are a critical tool in the delivery of genome modification reagents, CRISPR components (guide RNAs, Cas9 protein, and donor ...

wet-beveling-microinjection-needles-utilizing-constant-air-pressure

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284 Views.  University of Maryland. This protocol describes the assembly of a pneumatic system for the delivery of pressurized air to a needle during the process of needle beveling. The protocol further describes the beveling process for creating sharp microinjection needles and how to gauge the relative opening size of the needle.

Video: Author Spotlight: Enhancing Microinjection Needle Quality by Wet Beveling

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 ...

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 ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

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 ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.