Name: Video: Procedure for the Wet Beveling of Borosilicate Microinjection Needles Under Constant Delivery of Pressurized Air
Uploaded: 2024-09-27T13:20:00
Description: 32 Views. Procedure for the Wet Beveling of Borosilicate Microinjection Needles Under Constant Delivery of Pressurized Air

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

Protocol for Wet Beveling of Microinjection Needles Under Constant Air Pressure

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.

Author Spotlight: Enhancing Microinjection Needle Quality by Wet Beveling

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

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

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

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

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.