Name: Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture (Video) | JoVE Journal
Uploaded: 2008-02-13T11:00:00.000+00:00
Duration: 17 min 42 s
Description: Learn biolistic transfection of neurons in hippocampal slice cultures using DNA-coated gold bullets. This method enables targeted gene delivery for studying neuronal morphology with clean visualization.

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We would like to thank Mark Lucanic and Hwai-Jong Cheng for help with acquiring the low magnification images of entire hippocampal slices presented in this video. We would also like to thank Sarah Parrish for aiding with the text accompanying the video.

PROTOCOL FOR BIOLISTIC TRANSFECTION
Making bullets
Bullets are good for up to 6 months, but may begin to decrease in transfection efficiency after 2-3 months. Bullets should be stored at 4&deg;C in the presence of dessicant pellets. Always let the scintillation vials, in which bullets are stored, warm to room temperature before opening the vial.
Before getting started have ready:
<ul>
<li>Spermidine (0.05M )</li>
<li>CaCl2 (1M)</li>
<li>EtOH (100%-high grade-unopened bottle)</li>
<li>Autoclaved H20</li>
<li>DNA to be transfected</li>
<li>Polyvinylpyrrolidone (PVP-20 mg/ml stock)</li>
<li>2 X 15 ml conical (2/bullet set)</li>
<li>Tubing (cut into 1 X ~ 30 inches/ bullet set)</li>
<li>Scintillation vials (1/bullet set)</li>
<li>Desiccation pellets </li>
<li>10 ml syringe w/tubing on the end</li>
</ul>
Prepare tubing:
<ol>
<li>Dry ~ 30 inches of tubing (about 2 inches extending beyond right O-ring) in the tubing station w/ N2 gas (0.4 pressure) for a minimum of 20 min.</li>
</ol>
Precipitating DNA on gold beads:
<ol>
<li>Prepare DNA to be transfected in 50 &mu;mL total volume in microcentrifuge tube (maximum 50 &mu;g total DNA)</li>
<li>Weigh out 6-8 mg of gold and transfer to an microcentrifuge tube</li>
<li>Add 100 &mu;L of 0.05M spermidine to gold and sonicate for 20 sec</li>
<li>Add the 50 &mu;L of DNA to the gold/spermidine</li>
<li>Vortex (around setting 5) w/cap open</li>
<li>Add drop wise 100 &mu;L of 1M CaCl2</li>
<li>Sonicate briefly (&lt; 5 sec)</li>
<li>Allow to precipitate for 10 min at room temp</li>
<li>Sonicate briefly</li>
</ol>
Prepare PVP solution during sonication:
<ol>
<li>Make dilute PVP solution in 15 ml conical, 3.5mL dilute PVP for each set of bullets (add 7.5 &mu;L of 20mg/ml PVP stock to 3.5 ml 100 % EtOH)</li>
</ol>
Rinsing gold beads with EtOH:
<ol>
<li>Spin down gold beads at full speed for 10 sec in microcentrifuge</li>
<li>Discard supernatant, but keep a small amount of liquid on top of gold beads</li>
<li>Wash 3X with 1ml EtOH, sonicate briefly each time, if necessary</li>
</ol>
Resuspend gold/DNA in PVP solution:
<ol>
<li>Resuspend the gold pellet in 200 &mu;L of the 3.5 ml dilute PVP/EtOH solution</li>
<li>Vortex the gold pellet to resuspend (briefly sonicate if necessary)</li>
<li>Transfer the 200 &mu;L of gold/PVP solution to a new 15 mL conical</li>
<li>Continue in this way, adding 200 &mu;L of PVP at a time until all the gold has been transferred to the 15 mL conical, and the final volume is 3.2 mL</li>
</ol>
Coating tubing:
<ol>
<li>Turn off N2 at the tubing station and remove dried tubing</li>
<li>Attach dried tubing to the 10 ml syringe</li>
<li>Vigorously shake the 15 mL conical filled with gold/PVP</li>
<li>Place the end of the tubing in gold/PVP solution and suck up slowly being careful to avoid bubbles</li>
<li>Leave ~ 2 inches dry from both ends of the tubing</li>
<li>With the tubing still attached to syringe and filled with the gold/PVP solution, carefully place the tubing back into station</li>
<li>Mark the ends of the tubing where the solution sits</li>
<li>Let the gold settle for 5 min with the syringe attached</li>
<li>Suck-up the solution very slowly by pulling the syringe so that the settled gold is left behind (make sure not to back-wash)</li>
<li>Disconnect syringe</li>
<li>Rotate 90&deg; and let sit 2 seconds</li>
<li>Rotate 180&deg; and let sit another 2 seconds</li>
<li>Rotate tube for 5 sec</li>
<li>Turn on the N2 so that the valve reads between 0.4 - 0.5 and spin the gold-coated tubing while drying for 5 min.</li>
</ol>
Cutting tubing:
<ol>
<li>Remove the tubing from the tubing prep-station and cut off the ends at the marks.</li>
<li>Put a labeled scintillation vial under the tubing chopper to catch cut cartridges</li>
<li>Place tubing in the tubing chopper and cut the tubing with clean razor</li>
<li>Place a desiccation pellets in scintillation vial</li>
<li>Store cartridges at 4&deg;C</li>
</ol>
Shooting Tissue Slices
When shooting cultured slices, it is important to employ a relatively sterile technique in order to avoid contamination. Remember, that&nbsp;when shooting two different constructs, both sets of bullets can be loaded into the same cartridge holder, but the barrel-liner and screen should be changed between constructs.
Before getting started have ready:
<ul>
<li>DNA bullets (let scintillation vial warm to room temperature before opening)</li>
<li>Tissue slices</li>
<li>Helios Gene Gun </li>
<li>Helium tank with gene gun hose attached </li>
<li>Cartridge holder </li>
<li>Barrel liner with plastic O-ring in place </li>
<li>Diffuser (modified) </li>
<li>Diffuser Screen </li>
<li>Forceps </li>
</ul>
Prepping the gene gun:
<ol>
<li>Using forceps place the gold coated plastic cartridges into the cartridge holder.</li>
<li>Place the cartridge holder into the gene gun by first pushing back lock</li>
<li>Secure the cartridge holder with lock</li>
<li>Obtain a barrel liner with an O-ring and the diffuser screen securely in place</li>
<li>Screw the barrel-liner into the gene gun</li>
</ol>
Shooting cultured slices with the gene gun:
<ol>
<li>Put on ear muffs</li>
<li>Plug the gene gun into hose connected Helium gas tank</li>
<li>Turn up pressure on tank to 180 PSI</li>
<li>Shoot a blank into the air in order to remove any dust or debris from the diffuser screen. To shoot the gun, first depress the safety interlock button until the gun beeps, then pull the trigger.</li>
<li>After shooting the blank, remove the slices from the incubator</li>
<li>Advance gun to first construct</li>
<li>Shoot with the diffuser screen about 0.5 - 1 inch from the slice</li>
<li>Advance the cartridge between wells.</li>
<li>After shooting the last well, place the slices back into the incubator</li>
<li>Turn off gas and release pressure before detaching the gun from the Helium hose</li>
<li>Unscrew the barrel liner, and remove the cartridge and the used shells</li>
</ol>
Cleaning cartridge holders and barrel liners:
<ol>
<li>Place cartridges, barrel liners, and diffuser screens in a beaker with soapy water</li>
<li>Place beaker in bath sonicator and sonicate for 20 min</li>
<li>Thoroughly rinse with water until all soap residues are removed</li>
<li>Soak in 70% EtOH for an hour</li>
<li>Place on dry paper towel overnight to dry</li>
<li>Clean cartridges, barrel liners, and screens can then be reused in subsequent biolistic transfections</li>
</ol>
Trouble shooting tips for Biolistic Transfection
Biolistic transfection can sometimes result in suboptimal transfection conditions.&nbsp; It can lead to too few or too many cells transfected, too high or too low expression levels, or may cause tissue slices to become generally unhealthy. Often unhealthy slices result from the pressure blast. &nbsp;Here are some tips to obtain optimally transfected tissue slices.
If slices appear to be unhealthy (or if too many cells are transfected/ slice), try:
<ul>
<li>increasing shooting distance</li>
<li>decreasing pressure on gas tank</li>
<li>decreasing the amount of gold</li>
<li>cutting out the center of the Bio-Rad diffuser and replacing the center with a diffuser screen.</li>
</ul>
If too few cells are transfected, try:
<ul>
<li>decreasing shooting distance</li>
<li>increasing pressure on gas tank</li>
<li>increasing the amount of gold</li>
<li>increasing the number of tissue slices/ well</li>
<li>make sure the O-ring in the barrel-liner is intact.</li>
</ul>
If you obtain slices with both too many AND too few cells transfected during the same shooting make sure:
<ul>
<li>that you are holding the gun symmetrically above well</li>
<li>that gold is evenly coating the inside of tubing. (If you are getting a line of gold, draw out the supernatant from the tubing at a faster rate once the gold has settled and rotate the gold longer before turning on the nitrogen. If, on the other hand, you are getting streaking of gold, this is likely due to too much moisture exposure during bullet making: make sure you are using an unopened bottle of EtOH, that the tubing is thoroughly dry before coating, and that you capping lids and preparing bullets in a timely manner).</li>
</ul>
If transfection efficiency seems optimal (# cells transfected/ slice), but the expression level seems too high or too low:
<ul>
<li>adjust the ratio DNA to gold.&nbsp; (e.g., if it is too low maintain the amount of gold but increase the amount of DNA.)</li>
<li>adjust the amount of time between shooting and data collection</li>
</ul>
In the case of dual transfection, if you are getting too little expression of one of your constructs, adjust the ratio of the two constructs in the appropriate manner and make sure that both constructs are under the same promoter, so as to make sure that one promoter will not out-compete the other.

<ol><li>Klein, T. M., Fromm, M., Weissinger, A., Tomes, D., Schaaf, S., Sletten, M., Sanford, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transfer+of+foreign+genes+into+intact+maize+cells+with+high-velocity+microprojectiles%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Transfer of foreign genes into intact maize cells with high-velocity microprojectiles.</a> Proc. Natl. Acad. Sci. USA. 85, 4305-4309 (1988).</li>
<li>Wellmann, H., Kaltschmidt, B., Kaltschmidt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+protocol+for+biolistic+transfection+of+brain+slices+and+dissociated+cultured+neurons+with+a+hand-held+gene+gun%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">Optimized protocol for biolistic transfection of brain slices and dissociated cultured neurons with a hand-held gene gun.</a> J. Neurosci. Methods. 92, 55-64 (1999).</li>
<li>McAllister, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biolistic+transfection+of+neurons%5BTitle%5D)+AND+%22Sci.+STKE%22%5BJournal%5D)">Biolistic transfection of neurons.</a> Sci. STKE. 51, 1-13 (2000).</li>
<li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50, 823-839 (2006).</li>
</ol>

Biolistic transfection is a physical means of transfecting cells via bombardment of living tissue with high velocity DNA coated gold particles. In particle mediated gene transfer, in general transfected cells result when the bullet comes to rest in the nucleus. While many different transfection methods exist, such as microinjection, lipofection, calcium-phosphate transfection, electroporation and viral transfection, biolistic transfection can offer a less labor intensive, and efficient alternative for transfecting cells that are not easily transfected using these other methods. In fact, it was first developed as a technique for gene transfer in plants since the presence of the cell wall made transfection difficult using preexisting methods1. Similarly, biolistics has gained popularity in the field of neurobiology since post-mitotic neurons are notoriously difficult to transfect2,3. In particular, it is widely recognized as the principle technique to be used in experiments aimed at assessing fine morphology of single neurons in intact brain slices. The methods described here provide a detailed and comprehensive explanation of how to perform biolistic transfection of cultured brain slices using a hand held gene-gun (the Helios Gene Gun system; Bio-Rad).
Biolistic transfection has become the preferred method for transfecting neurons in slice culture because it allows transfection of a sparse number of cells throughout the brain slice. In this way, individual neurons can be examined in isolation. Since this technique is particularly well suited for transfecting neurons in brain slice, it is often used in conjunction with 2-photon microscopy, since 2-photon excitation enables visualization of fluorescently labeled cells deep within light scattering tissue4. Indeed the high magnification images of single pyramidal neurons presented throughout this video were captured using a custom built 2-photon laser scanning microscope. In conclusion biolistic transfection is an efficient means of transfecting individual cells within the context of a high density of surrounding cells, and as such has proven extremely useful for fluorescently labeling individual neurons in neuronal slice culture.

<table><tbody><tr><td>1.6um gold particles (microcarrier)</td><td>0.25g</td><td>Bio-Rad</td><td>1652264</td><td></td></tr><tr><td>1M CaCl2</td><td></td><td>Fluka</td><td>21115</td><td></td></tr><tr><td>100% EtOH</td><td>1pint</td><td>Goldshield</td><td></td><td></td></tr><tr><td>Cartridge Tubing</td><td></td><td>Bio-Rad</td><td>1652412</td><td></td></tr><tr><td>Scintillation vials</td><td>20ml, 500/case</td><td>VWR international</td><td>66021-668</td><td></td></tr><tr><td>Dessication pellets</td><td>&amp;#8221;Dricap&amp;#8221; </td><td>MTI (800-445-9890)</td><td></td><td></td></tr><tr><td>Water bath sonicator</td><td>model 50T</td><td>VWR international</td><td></td><td></td></tr><tr><td>Cartridge holder</td><td>pack of 5</td><td>Bio-Rad</td><td>1652426</td><td></td></tr><tr><td>Barrel liner</td><td>pack of 5</td><td>Bio-Rad</td><td>1652417</td><td></td></tr><tr><td>Diffuser screen</td><td>pack of 5</td><td>Bio-Rad</td><td>1652475</td><td></td></tr><tr><td>O-ring</td><td>75 Viton, Size 007, Qty, 100</td><td>McMaster-Carr</td><td></td><td></td></tr><tr><td>Screen (mesh)</td><td></td><td>McMaster-Carr</td><td>144258</td><td></td></tr><tr><td>PVP </td><td>20mg/ml in EtOH</td><td>Bio-Rad</td><td></td><td>Comes with tubing from biorad</td></tr><tr><td>Tubing prep station</td><td></td><td>Bio-Rad</td><td>1652418</td><td></td></tr><tr><td>Tubing cutter</td><td></td><td>Bio-Rad</td><td>1652422</td><td></td></tr><tr><td>Helios Gene-Gun</td><td></td><td>Bio-Rad</td><td>1652411</td><td></td></tr><tr><td>Gene gun hose </td><td></td><td>Bio-Rad</td><td></td><td>Comes with Gene-Gun</td></tr><tr><td>Spermidine</td><td>5g</td><td>Sigma-Aldrich</td><td>s-0266</td><td></td></tr><tr><td colspan="5">Although we have listed all the products seperately, it is more economical to purchase &quot;Helios Gene-Gun System&quot;, which includes with all the products from Bio-Rad listed above (cat # 165-2431).</td></tr></tbody></table>

preparation of gene gun bullets and biolistic transfection of neurons in slice culture

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

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.

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Source: Keener, D. G., et al. Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons. J. Vis. Exp. (2020).This video demonstrates the process of single-cell electroporation to introduce plasmid DNA into neurons within mouse hippocampal slice cultures. The procedure involves precise pipette positioning, pressure adjustments, and electroporation pulses to transiently permeabilize the neuronal membrane,...

Gene Expression via Single-Cell Electroporation in Mouse Hippocampal Slice Cultures

This video demonstrates the protocol for single-cell electroporation of genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice cultures. The technique is useful to examine specific molecular and physiological functions of neurons, including cell autonomous mechanisms and transsynaptic protein interactions.

Single Cell Electroporation in Organotypic Brain Slice Cultures: An In Vitro Technique to Deliver Plasmids Inside Targeted Neurons in Brain Hippocampal Slices

Source: Denaxa, M., el al., Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains. J. Vis. Exp. (2019)This video demonstrates the procedure for injecting a plasmid vector carrying the gene of interest into interneuron progenitors in an embryonic brain slice, followed by electroporation for plasmid entry and gene expression. These modified progenitors can later be used for treating brain disorders.

Agarose-Embedded Electroporation-Assisted Gene Transfer in Mouse Cortical Interneuron Progenitors

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Learn biolistic transfection of neurons in hippocampal slice cultures using DNA-coated gold bullets. This method enables targeted gene delivery for studying neuronal morphology with clean visualization.

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

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