In the first step of this injection procedure, a working concentration of DNA solution is centrifuge to sediment. Particulate matter injection pipettes fabricated from BO silicate glass capillary tubing are then filled with the DNA solution. Cells are placed on the microscope stage and the visualization of the cell nucleus is optimized.
The pipette is aligned with the nucleus and the cell is injected. Multiple cells are injected in each dish the following day. Successfully injected cells are identified by the expression of a reporter gene.
Hi, I'm Van Mu from the laboratory of Dr.Stephen Akeda at the National Institute on Alcohol Abuse and Alcoholism in the National Institutes of Health. I'm Damien Williams, also from the Akeda Lab. And I'm Eugene run from the AKEDA lab.
Today we'll show you the procedure for the INTERCLEAR micro injection of CDNA constructs into dissociated neurons. This is an effective method for hetero expressing proteins of interest in post mitotic cells, which are traditionally difficult to transfect. We use this technique in our lab to study the mechanisms underlying G protein modulation of N channels.
So let's get dark teeth. Begin by plating dissociated adult mammalian neurons on cell culture dishes coated with 0.1 milligram per milliliter of high molecular weight poly L lysine. This aids adhesion of neurons to the bottom surface of dishes.
Glass cloning cylinders can be used to concentrate plated neurons in a smaller, more confined area. To minimize the distance between neurons to be injected. Place dishes in a 37 degrees Celsius incubator for three to six hours before performing intranuclear micro injections to allow sufficient time for cells to adhere to the bottom of dishes.
While the neurons are incubating Microinjection pipettes are prepared using a Sutter P 97 flaming brown puller and thin walled boro silicate glass capillary tubes. We use a two stage pull program with the following settings for pull one and for pull two. Since the settings required vary with different batches of glass and by the condition of the heating filament in the puller, they should be adjusted as needed.
At least five injection pipettes should be pulled per dish of neurons in case the pipettes become damaged or there are problems during the injection session. The pulled injection pipettes should be stored in a covered container to prevent dust from entering the microinjection pipette tip immediately prior to injection plasmid CDNAs are diluted to the working concentration. Place a small piece of paraform on the work surface and remove the paper backing.
Add the CD NA stock solution and TE buffer to the param. To dilute the CD NA, we recommend using a final CDNA concentration of less than 200 nanograms per microliter and a total volume between two and 10 microliters. If the protein encoded by the CD NA is untagged a reporter plasmid encoding, EGFP, for example, can be co injected with the experimental CD NA.To confirm successful injection and expression, mix the CD NNA Solutions by pipetting up and down several times on perfil.
Being careful to avoid introducing air bubbles in order to sediment any particles in the cDNA solution that may later block the microinjection pipette. Use a micro loader pipette tip to transfer it to a prepared hematocrit tube which has been cleaned, cut and flame sealed at one end. Then place the hematocrit tube into a 1.5 milliliter micro centrifuge tube and spin for 15 to 30 minutes.
10, 000 GS at room temperature. Once the centrifugation is complete, set the tube containing the cDNA injection solution aside. The solution can be kept at room temperature during the injection session one to two hours.
After plating cells, remove the glass cloning cylinders. Then return the plated cells into the incubator for the remainder of the incubation period prior to the injection session. To start injecting, first, take a dish of plated neurons from the incubator and place it on the microscope stage of an injection setup.
The setup consists of an inverted phase contrast microscope, a pressure injector connected to a microm manipulator and a video monitoring system. Under phase contrast, neurons appear as spherical cells with a clearly visible nucleus as a central round organelle with a dark membrane and may contain a single or multiple nucleoli. Using a micro loader pipette tip, draw up two microliters of the prepared CD NA from the hematocrit tube.
Being careful to avoid the solution near the bottom of the tube where particles may have settled. Transfer the solution into a microinjection pipette. The filament in the microinjection pipette should aid in drawing the solution to the tip.
But if small air bubbles persist, gently flick the side of the glass to dislodge them, ensure that the capillary holder of the pressure injector is fixed at a 45 degree angle. Then insert the microinjection pipette into the capillary holder and secure the holder to the micro manipulator. Next, lower the microinjection pipette into the dish using the microm manipulator as the pipette enters the culture medium, a meniscus forms that alters the pathway of light through the specimen and thus reduces the image quality of the neurons.
To alleviate this problem, adjust the phasing turret until the nuclei of neurons are clearly visible. Before beginning injections. Use the clean function of the pressure injection system to clear the pipette of debris and to check the quality of microinjection pipettes.
Ejected solution should be visible on the screen. Upon using this function, the fluid from an optimally sized pipette tip is ejected as a thin stream. If the microinjection pipette is clogged or is too narrow, it will not eject fluid When the clean function is used.
If the tip is too wide or has been broken, fluid will be ejected from the tip as a broad stream. If the tip appears suboptimal during injections, replace the injection pipette now center the microinjection pipette in the viewing monitor. Keeping the nucleus in focus lower the tip of the pipette until it is in focus adjacent to a neuron.
Set this position as the lower Z axis limit on the micro manipulator. This position is the depth the microinjection pipette will advance to during injections. Next position the tip approximately 30 micrometers above this point.
So the microinjection pipette clears the top of neurons. Now program the settings of the microm manipulator for micro injections. Set the inject mode of an einor microm manipulator to on and the impale function to off to minimize cell damage.
During microinjection, designate a diagonal injection path by selecting the axial mode. Set the injection speed to 300 micrometers per second and synchronize the pressure buildup at the limit. Next program.
The settings of the pressure injector set the injection pressure or PI between 100 and 200 Hector Pascals or HPA, the injection duration or ti to 0.3 seconds and the compensation pressure or PC to 30 HPA. Once the manipulator settings have been adjusted, intranuclear injections can be performed. Using the microscope's XY axis stage control position, the neuron under the tip of the microinjection pipette.
Align the tip of the pipette above the center of the nucleus by focusing up and down between the tip and the nucleo lie. Inject the nucleus by pressing the inject button on the micro manipulator. The injection of the relatively low viscosity.
CDNA solution often appears as a white plume in the nucleus. When viewed under phase contrast optics. Continue injecting neurons by repositioning the microscope stage to align the next neuron under the microinjection pipette.
Systematically navigate through the dish. Continuing to inject nuclei. Use the clean function described previously every 10 to 15 injections to check the quality of the microinjection pipette.
If the pipette is clogged or damaged, replaced with a new injection. Pipette unevenness of the bottom surface of the tissue culture dishes can cause the injection pipette to miss the cell or prevent the white plume from being visible in the nucleus. In these cases, simply adjust the microinjection pipette tip to the same plane of focus as the nucleus, and set this as the new x-axis limit.
Then continue injecting neurons. Sometimes the microinjection pipette does not penetrate the nuclear membrane and simply nudges the nucleus. If this happens, 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. Once 50 to a hundred nuclei have been injected, return the dish of neurons to the incubator for overnight incubation. After 16 hours successfully injected neurons can be identified by expression of a reporter gene.
In this field, one superior cervical ganglion or SCG neuron has been injected and can be identified by expression of EGFP. Non injected SCG neurons, which do not have endogenous G protein coupled inwardly rectifying potassium or grk channels do not give rise to measurable currents during a voltage ramp. When exposed to the G-protein coupled receptor agonist norepinephrine by comparison, successful injection of plasmid CD NA encoding, a functional grk channel gives rise to large currents during a voltage ramp when exposed to norepinephrine.
The currents measured here are typical of Kirk channels and are characterized by activation by norepinephrine inward rectification and inhibition by the putative grk channel blocker. Barium, We've just shown you how to inject CD NA into the nucleus of neurons. When carrying out this technique, it's important to remember four things.
One, to spin the DNA before injection. Two, to make sure the nucleus is clearly visible. Three, to readjust the Z axis limit when navigating around the dish.
And four, to regularly check the quality of the pep tip between injections. So that's a thank you for watching and good luck with your experiment.
