Center for Neuroscience, University of California, Davis
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).
PROTOCOL FOR BIOLISTIC TRANSFECTION
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:
Precipitating DNA on gold beads:
Prepare PVP solution during sonication:
Rinsing gold beads with EtOH:
Resuspend gold/DNA in PVP solution:
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:
Prepping the gene gun:
Shooting cultured slices with the gene gun:
Cleaning cartridge holders and barrel liners:
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:
If too few cells are transfected, try:
If you obtain slices with both too many AND too few cells transfected during the same shooting make sure:
If transfection efficiency seems optimal (# cells transfected/ slice), but the expression level seems too high or too low:
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.
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.
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.
|1.6um gold particles (microcarrier)||0.25g||Bio-Rad||1652264|
|Scintillation vials||20ml, 500/case||VWR international||66021-668|
|Dessication pellets||€śDricap€ť||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|
|PVP||20mg/ml in EtOH||Bio-Rad||Comes with tubing from biorad|
|Tubing prep station||Bio-Rad||1652418|
|Gene gun hose||Bio-Rad||Comes with Gene-Gun|
|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).|
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).