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 ...

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>

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.

Author Spotlight: Enhancing Microinjection Needle Quality by Wet Beveling

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

The Insect Transformation Facility, we are a university service facility that helps clients make genetically modified insects. Their research ranges from neuroscience in which they are doing research on mosquito olfaction to public health research in which they are making vaccines to malaria, to expressing novel proteins within the black soldier fly. Traditionally, needles have been opened by pulling the needles and then brushing them against something in order to open that needle, and that can produce needles which are either perfect for microinjection or unusable.And so this method allows us to open the needle in a concise fashion in which we get sharp needles each time. This protocol gives feedback on the opening size of the needle. Once that needle is open, you can dial back the pressure that is being delivered to the tip of the needle, and the higher the pressure at which air bubbles stop flowing, the smaller the opening size of that needle.

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...

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.

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

Research

JoVE Journal

Biology

117 visualizações.  University of Maryland. A Insect Transformation Facility, somos uma instalação de serviços universitários que ajuda os clientes a produzir insetos geneticamente modificados. Suas pesquisas vão desde a neurociência, na qual eles estão fazendo pesquisas sobre o olfato do mosquito, até pesquisas de saúde pública, nas quais estão fazendo vacinas contra a malária, até a expressão de novas proteínas dentro da mosca soldado negra. Tradicionalmente, as agulhas são abertas pu...