procedure for the wet beveling of borosilicate microinjection needles under constant delivery of pressurized air

<meta charset="utf8"></head><body>Protocolo para chanfro úmido de agulhas de microinjeção sob pressão de ar constante

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.

<meta charset="utf8"></head><body>Autor em destaque: Melhorando a qualidade da agulha de microinjeção por chanfro úmido

Chanfro úmido de agulhas de microinjeção utilizando pressão de ar constante para feedback sobre a abertura da agulha

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.

Procedimento para o chanfro úmido de agulhas de microinjeção de borossilicato sob fornecimento constante de ar pressurizado

procedure-for-wet-beveling-borosilicate-microinjection-needles-under

Research

JoVE Journal

Biology

Procedure for the Wet Beveling of Borosilicate Microinjection Needles Under Constant Delivery of Pressurized Air

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.

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 ...

Biologia

Preparing the Grinding Assembly for the Wet Beveling of Microinjection Needles

Prepare the grinding assembly as per the manufacturer's guidelines, which includes setting up the abrasive plate and attaching the retaining ring equipped with magnets. Then apply 10 drops of pedestal oil onto the pedestals optically flat surface. Place the grinding assembly on the pedestal and initiate the grinding operation.Activate the light source to illuminate the work area, ensuring the light is behind the beveled edge, shining at an angle of 45 degrees to the abrasive plate and needle. Rotate the microscope head in place at 90x magnification. Briefly halt the spinning of the abrasive plate to focus the microscope on the surface of the plate.Add 1%wetting agent to the wick until the wick is completely wet. Then, add 1%wetting agent to the surface of the abrasive plate, ensuring it covers the surface without leaking onto the black retaining ring. Place the pre-wet wick onto the surface of the abrasive plate while it is spinning, ensuring the wick is on the left side of the plate while looking down from the top and stretches from 11 o'clock to six o'clock position with the plate as a clock face.Make sure the wick does not ride on the black portion of the retaining ring.

Preparando o conjunto de retificação para o chanfro úmido de agulhas de microinjeção

To begin beveling the borosilicate needle after preparing the grinding assembly, insert the needle into the needle holder and tighten the retaining ring to secure the needle in place. Open the regulator and increase the pressure to 24 pounds per square inch. Rotate the course adjustment knob enough to raise the needle holder till the needle is raised higher than the surface of the rotating abrasive plate.Rotate the entire manipulator so the needle swings into place above the rotating plate. Position the needle on the plate such that the rotation of the plate moves it away from the needle tip. Watching from the side, use the coarse adjustment knob to lower the needle toward the abrasive plate surface and stop when the needle is almost touching the surface of the liquid.Adjust the needle position so that it is in the center of the field of view. Once in the center, slowly increase the magnification, making slight adjustments to keep the needle tip in the center of view. At maximum magnification, stop the grinding plate and focus the microscope on the surface of the abrasive plate.Once the surface is in focus, immediately restart the plate rotation. Using the manipulator course adjustment knob, lower the needle toward the abrasive plate. In the field of view, observe the needle and the shadow of the needle.When the two are close to touching, switch to the fine adjustment knob. Continue lowering the needle until it touches its shadow. At this point, read the caliper and note the reading.This reading indicates the abrasive plate surface is at or below this caliper measurement. Allow the needle to remain at this caliper reading for five to 10 seconds. Using the manipulator fine adjustment knob, raise the needle, while ensuring it stays underneath the surface of the wetting agent.Stop the rotation of the abrasive plate briefly to observe whether bubbles escape from the needle tip. If bubbles are not present, move the needle back to the previous caliper reading. Lower it slightly.Take a new caliper reading and repeat the steps as demonstrated previously. If bubbles are seen, restart the abrasive plate to observe if bubble formation occurs while the plate is rotating. Once bubbles are observed with the abrasive plate not rotating, slowly decrease the air pressure by rotating the regulator adjustment knob.When the bubbles stop flowing from the tip of the needle, note the pressure. Then, increase the air pressure until bubbles start flowing again from the needle tip. Once the beveling is over, raise the needle above the abrasive plate, ensuring it is high enough not to hit anything as the entire manipulator is rotated away from the plate and microscope.Once the needle is in a position where it can be safely removed, lower the air pressure to zero. Remove the needle and place it in a needle storage box like a Petri dish until it is used.