JoVE   
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Biology

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Neuroscience

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Immunology and Infection

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Clinical and Translational Medicine

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Bioengineering

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Applied Physics

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Chemistry

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Behavior

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Environment

|   

JoVE Science Education

General Laboratory Techniques

You do not have subscription access to videos in this collection. Learn more about access.

Basic Methods in Cellular and Molecular Biology

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms I

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms II

You have trial access to videos in this collection until May 31, 2014.

 JoVE Biology

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

1, 1

1Center for Neuroscience, University of California, Davis

Article
    Downloads Comments Metrics
     

    Summary

    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.

    Date Published: 2/13/2008, Issue 12; doi: 10.3791/675

    Cite this Article

    Woods, G., Zito, K. Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture. J. Vis. Exp. (12), e675, doi:10.3791/675 (2008).

    Abstract

    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.

    Protocol

    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°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:

    • Spermidine (0.05M )
    • CaCl2 (1M)
    • EtOH (100%-high grade-unopened bottle)
    • Autoclaved H20
    • DNA to be transfected
    • Polyvinylpyrrolidone (PVP-20 mg/ml stock)
    • 2 X 15 ml conical (2/bullet set)
    • Tubing (cut into 1 X ~ 30 inches/ bullet set)
    • Scintillation vials (1/bullet set)
    • Desiccation pellets
    • 10 ml syringe w/tubing on the end

    Prepare tubing:

    1. 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.

    Precipitating DNA on gold beads:

    1. Prepare DNA to be transfected in 50 μmL total volume in microcentrifuge tube (maximum 50 μg total DNA)
    2. Weigh out 6-8 mg of gold and transfer to an microcentrifuge tube
    3. Add 100 μL of 0.05M spermidine to gold and sonicate for 20 sec
    4. Add the 50 μL of DNA to the gold/spermidine
    5. Vortex (around setting 5) w/cap open
    6. Add drop wise 100 μL of 1M CaCl2
    7. Sonicate briefly (< 5 sec)
    8. Allow to precipitate for 10 min at room temp
    9. Sonicate briefly

    Prepare PVP solution during sonication:

    1. Make dilute PVP solution in 15 ml conical, 3.5mL dilute PVP for each set of bullets (add 7.5 μL of 20mg/ml PVP stock to 3.5 ml 100 % EtOH)

    Rinsing gold beads with EtOH:

    1. Spin down gold beads at full speed for 10 sec in microcentrifuge
    2. Discard supernatant, but keep a small amount of liquid on top of gold beads
    3. Wash 3X with 1ml EtOH, sonicate briefly each time, if necessary

    Resuspend gold/DNA in PVP solution:

    1. Resuspend the gold pellet in 200 μL of the 3.5 ml dilute PVP/EtOH solution
    2. Vortex the gold pellet to resuspend (briefly sonicate if necessary)
    3. Transfer the 200 μL of gold/PVP solution to a new 15 mL conical
    4. Continue in this way, adding 200 μL 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

    Coating tubing:

    1. Turn off N2 at the tubing station and remove dried tubing
    2. Attach dried tubing to the 10 ml syringe
    3. Vigorously shake the 15 mL conical filled with gold/PVP
    4. Place the end of the tubing in gold/PVP solution and suck up slowly being careful to avoid bubbles
    5. Leave ~ 2 inches dry from both ends of the tubing
    6. With the tubing still attached to syringe and filled with the gold/PVP solution, carefully place the tubing back into station
    7. Mark the ends of the tubing where the solution sits
    8. Let the gold settle for 5 min with the syringe attached
    9. Suck-up the solution very slowly by pulling the syringe so that the settled gold is left behind (make sure not to back-wash)
    10. Disconnect syringe
    11. Rotate 90° and let sit 2 seconds
    12. Rotate 180° and let sit another 2 seconds
    13. Rotate tube for 5 sec
    14. 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.

    Cutting tubing:

    1. Remove the tubing from the tubing prep-station and cut off the ends at the marks.
    2. Put a labeled scintillation vial under the tubing chopper to catch cut cartridges
    3. Place tubing in the tubing chopper and cut the tubing with clean razor
    4. Place a desiccation pellets in scintillation vial
    5. Store cartridges at 4°C

    Shooting Tissue Slices

    When shooting cultured slices, it is important to employ a relatively sterile technique in order to avoid contamination. Remember, that 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:

    • DNA bullets (let scintillation vial warm to room temperature before opening)
    • Tissue slices
    • Helios Gene Gun
    • Helium tank with gene gun hose attached
    • Cartridge holder
    • Barrel liner with plastic O-ring in place
    • Diffuser (modified)
    • Diffuser Screen
    • Forceps

    Prepping the gene gun:

    1. Using forceps place the gold coated plastic cartridges into the cartridge holder.
    2. Place the cartridge holder into the gene gun by first pushing back lock
    3. Secure the cartridge holder with lock
    4. Obtain a barrel liner with an O-ring and the diffuser screen securely in place
    5. Screw the barrel-liner into the gene gun

    Shooting cultured slices with the gene gun:

    1. Put on ear muffs
    2. Plug the gene gun into hose connected Helium gas tank
    3. Turn up pressure on tank to 180 PSI
    4. 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.
    5. After shooting the blank, remove the slices from the incubator
    6. Advance gun to first construct
    7. Shoot with the diffuser screen about 0.5 - 1 inch from the slice
    8. Advance the cartridge between wells.
    9. After shooting the last well, place the slices back into the incubator
    10. Turn off gas and release pressure before detaching the gun from the Helium hose
    11. Unscrew the barrel liner, and remove the cartridge and the used shells

    Cleaning cartridge holders and barrel liners:

    1. Place cartridges, barrel liners, and diffuser screens in a beaker with soapy water
    2. Place beaker in bath sonicator and sonicate for 20 min
    3. Thoroughly rinse with water until all soap residues are removed
    4. Soak in 70% EtOH for an hour
    5. Place on dry paper towel overnight to dry
    6. Clean cartridges, barrel liners, and screens can then be reused in subsequent biolistic transfections

    Trouble shooting tips for Biolistic Transfection

    Biolistic transfection can sometimes result in suboptimal transfection conditions.  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.  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:

    • increasing shooting distance
    • decreasing pressure on gas tank
    • decreasing the amount of gold
    • cutting out the center of the Bio-Rad diffuser and replacing the center with a diffuser screen.

    If too few cells are transfected, try:

    • decreasing shooting distance
    • increasing pressure on gas tank
    • increasing the amount of gold
    • increasing the number of tissue slices/ well
    • make sure the O-ring in the barrel-liner is intact.

    If you obtain slices with both too many AND too few cells transfected during the same shooting make sure:

    • that you are holding the gun symmetrically above well
    • 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).

    If transfection efficiency seems optimal (# cells transfected/ slice), but the expression level seems too high or too low:

    • adjust the ratio DNA to gold.  (e.g., if it is too low maintain the amount of gold but increase the amount of DNA.)
    • adjust the amount of time between shooting and data collection

    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.

    Discussion

    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.

    Disclosures

    Acknowledgements

    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.

    Materials

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

    References

    1. Klein, TM., Fromm, M., Weissinger, A., Tomes, D., Schaaf, S., Sletten, M. and Sanford, JC. Transfer of foreign genes into intact maize cells with high-velocity microprojectiles. Proc. Natl. Acad. Sci. USA. 85, 4305-4309 (1988).

    2. Wellmann, H., Kaltschmidt, B. and Kaltschmidt, C. Optimized protocol for biolistic transfection of brain slices and dissociated cultured neurons with a hand-held gene gun. J. Neurosci. Methods. 92, 55-64 (1999).

    3. McAllister, AK. Biolistic transfection of neurons. Sci. STKE 51, 1-13 (2000).

    4. Svoboda, K. and Yasuda, R. Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron. 50, 823-839 (2006).

    Comments

    19 Comments

    Wow! This is a great article! A detailed visual and written protocol, superb. Congratulations to the authors and editors!I have only one minor criticism: the use of "inches" (instead of centimeters).
    Reply

    Posted by: AnonymousFebruary 15, 2008, 7:38 AM

    Do you routinely have problems with humidity in the lab environment?  I work down at UTMB in Galveston, TX and we were told that it would be difficult to make the bullets because of our high relative humidity.  Your article is great, very concise and easy to follow, and it seems like you don't worry about relative humidity at all.Thanks for any help you can give!
    Reply

    Posted by: AnonymousFebruary 21, 2008, 3:46 PM

    It is relatively dry in Davis (~²0% humidity), so yes: we do not worry about humidity. But Dr. Zito used to make bullets back at Cold Spring Harbor in the thick of humid New York summers and never had any problems. If you dry your tubing thoroughly (~30 min) with nitrogen and use a newly unopened bottle of EtOH every time I imagine that you should be fine.-Georgia
    Reply

    Posted by: AnonymousFebruary 22, 2008, 2:29 PM

     First of all, I must credit the authors and video team for an extremely well written protocol.   I have been struggling to get a complete picture of biolistic tranformation and this video entry very effectively demonstrates the use of the Bio-Rad Gene Gun. I am in the process of developing a biolistic method for the transformation of bacterial cell cultures.  Since biolistics is not very often utilized for bacterial transformation even the technical reps over at Bio-Rad knew little about the details.  Do the authors or anybody else know others that have worked with transforming bacterial cell lines using the Gene Gun?  If so, I would appreciate any references for these methods.I am aware that their is a secodary biolistic system available through Bio-Rad called the method using a Biolistic® PDS-1000/He
    Particle Delivery System
    .  This system consists of a pressurized chamber component - as opposed to the Gene Gun - for particle delivery.  It is my understanding that this system is better for bacterial transformation.  However, I have come across a loaner Gene Gun system (not the PDS-1000) and would like to attempt bacterial transformation with this. Any help will be appreciated.      
    Reply

    Posted by: AnonymousFebruary 28, 2008, 2:20 PM

    Hi Trook, I'm interested to know if you succeeded in transforming bacteria with the gene gun?
    Reply

    Posted by: AnonymousAugust 3, 2011, 2:46 AM

    This is a wounderful video on use of gene gun. I would like to download this video, how could i should do this.  i am starting to apply this technique in studying ion channels.  could you please let me know the way of downloading this clip. Thanking you Sincerely Nagesh. M  
    Reply

    Posted by: AnonymousMay 16, 2008, 3:04 PM

    Greetings Nagesh!  Good to hear you want to put biolistic transfection to such noble use!  I spent my graduate career as a channelologist.  Please contact me directly and I will set you up with a way to access this video for download. AaronK@JoVE.com  
    Reply

    Posted by: Aaron K.May 16, 2008, 6:20 PM

    I had never done this protocol before, and using this technique has been HUGELY helpful.  It it an easy to use protocol, well written, and the video showing the steps have been so easy to follow. Thanks so much, with this protocol I am already becoming quite proficient, and my boss is thrilled with me.
    Reply

    Posted by: AnonymousJuly 2, 2008, 10:39 AM

    why do you use gold particles instead of tugstene particles?
    Reply

    Posted by: AnonymousOctober 18, 2008, 8:00 PM

    It turns out that tungsten can be toxic to cells, and catalytically
    degrades DNA.(Sanford et al. 1993). Hope that helps.
    Reply

    Posted by: AnonymousOctober 21, 2008, 12:49 PM

    Thank you very much for making this video! Very precise and easy to follow. This really helps my experiment.
    Reply

    Posted by: AnonymousNovember 29, 2008, 3:48 PM

    Hi...
    This protocol was very useful and I agree with all the positive comments posted above! I am using it to transect hippocampal brain slices.
    I have a question about the distribution of the bullets into the slices. Although, I am following this protocol, I am not able to achieve optimal transfection efficiency. Also, the distribution of the transfected cells throughout the slice dŒs not remain uniform. I have tried using the mesh suggested in this protocol and that too dŒs not make much difference; While In the images displayed in this protocol I could see very well uniformly transfected cells throughout the slice. Can you give any suggestions to improve uniformity in distribution of transfected cells?
    Reply

    Posted by: AnonymousJuly 14, 2010, 5:04 PM

    Hi Shruti,
    I can think of two likely possibilities:
    (1) the gold in the bullets is not evenly distributed -- try to make sure that it dŒs not end up all on one side
    (²) you are shooting too close to your samples-- try backing up a bit
    Best of luck!
    Reply

    Posted by: Karen Z.July 27, 2010, 12:15 PM

    how can i download the video?.
    Reply

    Posted by: AnonymousOctober 18, 2011, 6:54 AM

    It is not possible to download any of our video articles. This particular article is free to view so you can enjoy it many times over.

    Best,
    JoVE
    Reply

    Posted by: AnonymousOctober 18, 2011, 11:59 AM

    Hi, I was wondering if it is possible to stain the tissues with other antibodies either before or after labeling with the gene gun. I usually label brain slices with DiI using the gene gun but I also want to look at some other proteins and I was thinking of using the same slices for IHC as well if possible?
    Reply

    Posted by: AnonymousJanuary 26, 2012, 5:18 PM

    Hi Harpreet,
    We have never done this but I don't see why not... try it?
    Best of luck!
    Reply

    Posted by: AnonymousJanuary 29, 2012, 8:03 PM

    Dear Dr. Woods,

    First, I wanted to thank you for this video. It allows to show very clearly all stages of the biolistic transfection protocol.

    I would like to try your modified diffusion screen but the catalog number 144²58 is unobtainable.

    I would be very grateful if you could maybe give me the characteristics of your screen mesh please?

    I am looking forward to hearing from you.

    Best regards,

    Paula
    Reply

    Posted by: Paula P.July 6, 2012, 5:55 AM

    Hi Paula- sorry for the delay. The mesh can be obtained from McMaster-Carr - item number 9317T109 - http://www.mcmaster.com/#catalog/118/41²/=iirztc - Corrosion-Resistant 304 SS Wire Cloth Disc 100 X 100 Mesh, 1-1/4" Diameter, .0045" Wire Dia - let me know if you have any problems and good luck!
    Reply

    Posted by: Karen Z.July 22, 2012, 5:43 PM

    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Metrics

    Waiting
    simple hit counter