Making gene gun bullets (GOLD)
<ol>
<li>Make 100X PVP (0.5mg/ml) in 100% EtOH.</li>
<li>Weigh out 30mg of BioRad 1.6 Micron microcarrier gold and place it into a 1.5 ml eppendorf tube. </li>
<li>Calculate the amount of plasmid needed to give a concentration of 0.5-3 &micro;g plasmid /mg gold, and place the plasmid into the tube with the gold microcarriers. Combine several plasmids if needed.</li>
<li>Bring volume of plasmid to 100 &micro;l with distilled water.</li>
<li>Add 100 &micro;l of 0.05M spermidine into the tube...

<ol>
	<li>Landreth, . B. W., Agranoff, N. R., Smalheiser, S. M., Crain, M. B., Bornstein, R., Adler, P. J., Magistretti, A. G., Hyndman, W. J., Shoemaker, H., Rehm, T., Schafer, H., Betz, S. U., Kim, H., Takahashi, A. R., Caffe, A., Soderpalm, T., van Veen, H. C., Wong, S., Thompson, D. Y., Yu, S. J., Cringle, V. A., Alder, S. J., Taylor, G. M., Seigel, A. R., Caffe, P., Ahuja, B., Holmqvist, S., Azadi, J., Forsell, I., Holmqvist, J., Hatakeyama, R., Kageyama, S. S., Zhang, X. Y., Fu, C. J., Barnstable, S. W., Wang, X., Mu, W. J., Bowers, W. H., Klein, T., Inoue, M., Hojo, Y., Bessho, Y., Tano, J., Lee, R., Kageyama, A., Kretz, S. H., Hermening , S., Isenmann , I., Liljekvist-Larsson, K., Johansson, B., Reidel, W., Orisme, T., Goldmann, W. C., Smith, U., Wolfrum, H., Xin, J. A., Yannazzo, R. S., Duncan, E. V., Gregg, M., Singh, P., Koulen, S. A., Johnston, D. C., Tang, D. C., Lo, A. K., McAllister, K. a. t. z., LC, L. E., Rajagopalan, J. S., Malter, A. K., McAllister, W. -. B., Gan, J., Grutzendler, W., Wong, R., Wong, J., Lichtman, H., Sato, S., Hattori, S., Kawamoto, I., Kudoh, A., Hayashi, I., Yamamoto, G., Zhang, M. E., Selzer, R. L., Sohn, M. T., Murray, K., Schwarz, J., Nyitray, P., Purray, A. P., Franko, J. A., O'Brien, M., Holt, G., Whiteside, S. C., Lummis, M. H., Hastings, L., Klimaschewski, W., Nindl, M., Pimpl, P., Waltinger, K., Pfaller, P., Kettunen, J., Demas, C., Lohmann, N., Kasthuri, Y., Gong, R. O., Wong, N., Gamper, M. S., Shapiro, M. J., Wirth, P., Wahle, J., O'Brien, S. C., Lummis, A. M., Benediktsson, S. J., Schachtele, S. H., Green, M. E., Dailey, L., Chow, E. M., Levine, T. A., Reh, R. L., Rockhill, F. J., Daly, M. A., MacNeil, S. P., Brown, R. H., Masland, W., Sun, N., Li, S., He, W., Sun, N., Li, S., He, R. C., Stacy, R. O., Wong, E., Strettoi, V., Pignatelli, C., Rossi, V., Porciatti, B., Falsini, V., Pignatelli, E., Strettoi, V. P., Connaughton, D., Graham, R., Nelson, P. H., Edqvist, F., Hallbook, T. C., Jakobs, R. T., Libby, Y., Ben, S. W., John, R. H., Masland, Y., Qin, G., Xu, W., Wang, R., Roizenblatt, J. D., Weiland, S., Carcieri, G., Qiu, M., Behrend, M. S., Humayun, A., Koizumi, G., Zeck, Y., Ben, R. H., Masland, T. C., Jakobs, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explant+culture+of+adult+goldfish+retina:+a+model+for+the+study+of+CNS+regeneration.">Explant culture of adult goldfish retina: a model for the study of CNS regeneration.</a> Brain Res. 161, 39-55 (1979).</li></ol>

1. What can I do with this method?
<ul>
<li>See cell morphology clearly.</li>
<li>Label cells to record from them.</li>
<li>Overexpress proteins, like PSD95, but also other proteins that
modulate the cell's response, e.g. ion channels, receptors.</li>
<li>Express inhibitory RNAs.</li>
</ul>
2. How long can I keep adult retina?
<ul>
<li>4 days is no problem for adult rabbit. After six days the cells
look "sick", i.e. the axons retract, you see swelling of dendrites
and the recor...

organotypic culture of adult rabbit retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

Watch this Scientific Journal Video about Organotypic Culture of Adult Rabbit Retina at JoVE.com

Organotypic Culture of Adult Rabbit Retina

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

organotypic-culture-of-adult-rabbit-retina

Research

JoVE Journal

Biology

11.9K visualizações.  MGH - Massachusetts General Hospital. Este artigo demonstra a dissecção e incubação de retina de coelho e de partículas mediado por transferência de genes de codificação de plasmídeos GFP ou uma variedade de marcadores subcelular em células ganglionares da retina.