遺伝子銃の箇条書きとスライス培養における神経細胞の微粒子銃トランスフェクションの準備

Biotic Or gene gun Transfection is a physical means of transecting cells by bombarding tissue with high velocity DNA coated particles. This technique is an efficient and easy means of transecting neurons and is especially useful for transecting only a small number of cells in tissue slice. For example, in fluorescent microscopy, studies aimed to examine neuronal morphology in an intact slice.

Bic transfection facilitates visualization of individual neurons in a clean background of non transfected cells. In this video, we demonstrate a method for making DNA coated gene gun bullets and then performing Bic transfection on organotypic hippocampal slice cultures. Hi, I'm Georgia Woods.

I'm a graduate student in the laboratory of Dr.Karen Zito at the University of California at Davis. Today I will demonstrate how to make DNA coated bullets and how to perform Bic transfection on rat hippocampal slices. The basic steps underlying Bic transfection are to one coat gold particles with the DNA to be transfected two coat the inside of the plastic tubing with this gold three.

Cut the tubing into cartridges to load into the gene gun and four, shoot the tissue to be transfected. So let's get started. So Before we are ready to transfect, we first need to prepare the DNA coated bullets.

This will entail coating 1.6 micron gold particles with the DNA plasmid to be transfected. During shooting, an individual gold particle will act as the bullet that will penetrate the cell leading to transfection. So first, we need to prepare the DNA to be transfected.

Ideally, you want to use less than 50 micrograms of DNA per set of bullets in a total volume of 50 microliters of water. Keep in mind that the exact amount of DNA to be used should be determined empirically for each construct based on obtaining the optimal expression level. Now that we have our DNA aliquot, we need to add sperming to the gold beads.

Add to the eight mgs of pre weighed gold, a hundred microliters of sperm, meine and sonicate. Around 20 seconds. It's sperm.

Meine is a poly amine which positively charges the gold leads to negatively charged DNA interacting with the positively charged metal. After the 22nd son, add the 50 microliter DNA water solution to the gold. Once the DNA has been added to the beads vortex while adding a hundred microliters of calcium chloride dropwise calcium chloride causes the DNA to precipitate.

After a brief vortex allow solution to precipitate for 10 minutes. After the 10 minute precipitation spin DNA and gold full speed for 30 seconds in a tabletop centrifuge after the 32nd high speed spin pipette off the majority of the supernatant, but avoid sucking up the gold pellet. At this point, I'm just removing any DNA, which has not precipitated on the gold.

The gold particles are then washed in one mill of brand new ethanol and sonicated.Briefly. Once the DNA precipitates, it tends to cause the gold to clump A light sonication breaks up these gold clumps. But be careful not to shear the DNA by overs sonication.

The gold pellets are spun down again and the ethanol is removed by pipetting. Repeat the ethanol wash two more times. After the third ethanol wash, add 200 microliters of the diluted poly vinyl perone or PVP solution and sonicate.

Briefly transfer over the coated gold to a 15 mil conical already containing 200 microliters of diluted PVP solution. PVP is a solvent that will cause the gold particles to adhere to the plastic tubing used. In the next step, remove traces of gold beads remaining in the epit tube by adding 200 microliters of PVP to the epi tube.

Then briefly fornicating and transferring over to the 15 mil conical. Repeat this until all the gold has been transferred over to the 15 mil conical. Transfer over the remaining PVP solution so that the gold is now resuspended in 3.2 mil solution of PVP ethanol.

Now we're ready to coat the plastic tubing that will be used to house the gene gun bullets when they're loaded into the gene gun. Now That the gold is coated with DNA, we need to line the inside of the plastic tubing with the gold particles. This gold coated plastic tubing is what will actually be loaded into the gene gun tub.

Begin, cut a piece of tubing so that it extends about two inches past the O ring. Once the tubing has been placed in the tubing prep station, turn the pressure valve so that the flow meter reads about 0.4. Nitrogen provides a water free atmosphere to dry the tubing, dry the tubing for about 20 minutes.

It is best to perform this drying step while you are coating the gold bullets with DNA as shown in the previous step. That way the tubing will be dry and will be ready to coat once you're done. Preparing the gold beads now that the tubing is dry and that the gold has been coated with the DNA.

Turn off the N two station before filling the tubing with your gold PVP solution and remove dried tubing from the station and attach to 10 mil syringe before sucking up the PVP and gold into the tubing. Thoroughly shake up the solution and immediately suck it up into the dried tubing, avoiding bubbles. Also, be careful not to suck up the solution into the syringe.

With the tubing now filled with the PVP Gold solution, carefully place the tubing back into the N two station. Let the solution settle for five minutes with the syringe attached so that the gold particles settle to the bottom of the tubing. Mark the ends of the tubing where the solution settles.

After the solution settles, a gold streak will be visible on the bottom of the tubing using the attached syringe. Pull off the supernatant very slowly, making sure not to backwash disconnect the tubing. Now we need to rotate and dry the tubing in order to adhere the gold evenly throughout the inside of the tubing.

First, spin the tubing 90 degrees. Let's settle two seconds. Now rotate another 180 degrees, and let's settle another two seconds.

Rotate five seconds and finally turn on the N two so that the station reads between 0.4 and 0.5 and spin while the tubing is drying for five minutes. Now that the five minutes are up and our gold is dry, we need to cut the tubing into cartridges that can be loaded into the gene gun. Now I will cut the tubing into about a half inch sections to make the shells that will be an appropriate size to load into the cylinder of the gene gun.

In order to collect the shells place seal simulation bile under the cutter, cut the tubing at the markings, cut the tubing with a clean razor. Place the tubing in the tubing chopper to cut an appropriate length. Simply feed the tubing forward until you feel resistance and then cut.

Once the tubing has been cut and collected in the scintillation vial place a desiccation pellet in the scintillation vial. Bullets are good for up to six months, but after two to three months may begin to show a decrease in transfection efficiency. So it is good practice to label the vial with the construct name and date.

Bullets can be used immediately after they are made or can be stored at four degrees Celsius. If you are using bullets stored at four degrees Celsius, let the sation vial warm at room temperature for about 15 minutes before opening in order to prevent the bullets from absorbing moisture. We are now ready to shoot the DNA bullets using a gene gun.

So Now that the bullets are made, I'm ready to actually bally transfect cells. I will be using a BioRad Helios gene gun and will be shooting rat hippocampal slices that have been incubated for five days since they were dissected. The gene gun will be connected to a helium gas tank.

When the gun is fired, a short burst of pressure from the tank will accelerate the gold particles at the slice. In general. When a gold particle ends up embedded in the cell's nucleus, then that cell will be become transfected.

Since I am transecting cultured slices, I employ a relatively sterile technique throughout Bic transfection in order to avoid contamination. Once the cartridges have warmed to room temperature, open the simulation vial and place the gold coated plastic cartridges into the cartridge holder. Then place the cartridge holder into the gene gun by first pushing back the cylinder lock.

Now the barrel liner needs to be attached to the gun. First, make sure the O-ring is in the barrel liner and that the diffuser screen is secure. The screen acts to spread the pressure from the barrel and leads to the spray of the gold for a more even dispersion of gold in the well.

Screw the barrel liner all the way into the gene gun. When shooting two different constructs, both sets of bullets can be loaded the same cartridge holder, but the barrel liner and screen should be changed between constructs. The gene gun is now ready to be attached to the helium source.

Plug the gene gun into the hose connected to the helium gas tank. It should snap into place and turn up the pressure on the tank to 180 PSI. The gun is now ready to shoot, but first, remember to wear ear protection and in order to remove any dust or debris from the diffuser screen, shoot a blank to shoot the gun.

First, press the safety interlock button until the gun beeps and pull the trigger. Now we're ready to shoot the slice. After shooting the blank, remove the slices from the incubator.

During our hippocampal dissections, we place four slices per well and arrange them so that they form the corners of a two centimeter square near the center of the membrane. Shoot each well with the diffuser screen about one half to one inch from the slice. Transfected cells can usually be examined within one to five days, depending on the construct.

Once all the wells have been shot, turn off the gas and release the pressure before detaching the gun from the helium hose. Remove the cartridges and the used shells. Unscrew the barrel liner, the cartridges, barrel liners, and the screens can be cleaned by placing them in a soapy water bath and sonic skating for 20 minutes and thoroughly rinsing in water and soaking in 70%ethanol for an hour and then placing them on a dry paper towel overnight.

Clean cartridges and screens can then be reused in subsequent biotic transfect. Now let's have a look at some of the transfection results. Even For a standard hippocampal slice preparation, there can be a fairly wide range of transfection efficiencies depending on the bullets and the slices.

Generally, a relatively low transfection efficiency is desirable in order to obtain nicely isolated transfected neurons. In addition, when too many cells are transfected, the overall health of the slice is compromised with optimal conditions. Usually less than 20 cells are per slice, but often other slices in the same well will end up with many more cells.

Transfected and other slices will end up with less cells. Transfected inconsistencies in transfection efficiency between slices is usually due to the fact that either the placement of the slices relative to the barrel was not symmetric, or the gold was not evenly coated around the tubing, but was concentrated on one side. If too many cells are transfected, try increasing the shooting distance from the sample or decreasing the shooting pressure or decreasing the amount of gold for making the bullets.

Here you can see a great example of transfected ca one parametal neuron On this particular cell, we can see a gold bullet embedded into the cell. Here's a closer look at a transfected neuron. In ca one, Sometimes Shooting can lead to unhealthy slices.

Often unhealthy slices result from the pressure blast. The Helios gene gun system comes with the diffuser with a metal cross at its center. We cut out the inner metal cross and instead use a metal screen in place of this cross.

We simply cut the screen a little larger than the end of the barrel liner and pinch it between the end of the barrel liner and the cut diffuser. We have found that this improves the health of slices. Additionally, optimal shooting conditions will vary with the experimental preparation in order to optimize for different experimental preparations, vary the pressure on the helium tank and the shooting distance from the sample.

Finally, in the event that you want to quote bullets with two different constructs for dual transfection, make sure that both constructs are under the same promoter. That way one promoter will not out-compete the other. I've Just shown you how to prepare gene gun bullets and how to transfect hippocampal slices using BIC transfection.

This is an extremely powerful method for performing morphological studies on single neurons that can be imaged in isolation. So that's it. Thanks for Watching and good luck with your experiments.