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. Vortex tube 30 seconds.</li>
<li>Vortex 1 minute at full speed. Sonicate for 3-5 minutes..</li>
<li>Dry an appropriate length of Tefzel tubing in a Biorad PDS-1000/He apparatus for 15 minutes.</li>
<li>After sonication, vortex for 1-2 minutes at full speed. Reduce speed slowly to allow the tube to be opened while vortexing.</li>
<li>Add 100 &micro;l of 1 M CaCl2 slowly to the tube drop wise while vortexing.</li>
<li>Leave tube at room temperature for 10 minutes.</li>
<li>Make 5 Micron filter. <ol>
<li>Take two 5ml syringes without needles.</li>
<li>Remove the plunger, and detach the black caps. Taking small scissors, cut off a part of the black cap to give a ring. Repeat on the other plunger to net two rings.</li>
<li>Sandwich a 5 Micron (Small Parts, Inc. nylon filter, &frac12; inch diameter) filter between the two rings, and push the sandwich into the tip of syringe. </li>
<li>Dangle syringe over a 50ml conical flask.</li>
</ol></li>
<li>After 10 minutes, vortex the tube for 30 seconds.</li>
<li>Centrifuge the tube for 30 seconds and save the pellet.</li>
<li>Add 1ml of 100% EtOH to pellet. Use pipette tip to break up the pellet, and vortex 30 seconds. Spin 30 seconds and remove EtOH. Repeat 2 more times.</li>
<li>Add 1ml of 100% EtOH to the pellet (break up with tip). Place this into the 5 Micron filter and let it flow through by gravity into the 50ml conical tube. Use more EtOH as needed to wash out tube or help with flow through.</li>
<li>Spin the 50ml tube at 2000rpm in centrifuge for 5-10 minutes. Remove supernatant, save pellet.</li>
<li>Depending on the desired density of microcarriers needed, add 800&micro;l-1.5ml 100% EtOH and corresponding 8 &micro;l-15 &micro;l PVP solution.</li>
<li>Swirl and immediately go to the Biorad PDS-1000/He apparatus.</li>
<li>Biorad PDS-1000/He apparatus <ol>
<li>Disconnect the tube connecting the N2 gas tank to the drying tube</li>
<li>Take out drying Tefzel tubing from machine</li>
<li>Insert one end into the 50ml conical tube, and attach a syringe to the other end of the tubing. Suck brown slurry into the middle of the tube.</li>
<li>Insert tubing back into machine.</li>
<li>Wait (without rotation) for 4 minutes</li>
<li>After 4 minutes, suck off EtOH slowly and carefully, making sure not to disturb microcarriers.</li>
<li>Rotate the tubing for 1 minute.</li>
<li>After 1 minute, connect the tube connecting the N2 gas tank to the drying tube and let tubing dry for 15 minutes.</li>
</ol></li>
<li>After 15 minutes, cut the tubing to bullet size to be used immediately or stored at 4&deg;C in an exsiccator. Bullets can be stored up to 3 months.</li>
</ol>
Preparation of gene-gun
<ol>
<li>Place bullets into a cartridge. Each piece of retina can be shot with 1-3 bullets.</li>
<li>Insert the cartridge into the gene-gun and attach the gun to a helium source. Set the pressure to 110 Psi.</li>
</ol>
Preparation of media
<ol>
<li>Dissection medium 
<table style="width: 229px; height: 157px;" border="0">
<tbody>
<tr>
<td>&nbsp;1 bottle Ames solution (Sigma, 8.8g) </td>
</tr>
<tr>
<td>&nbsp;1.9 gm sodium bicarbonate</td>
</tr>
<tr>
<td>&nbsp;10 ml 1% pen/strep/L-Glutamine (1:100) (Gibco/Invitrogen)</td>
</tr>
<tr>
<td>&nbsp;990 ml distilled water to 1L</td>
</tr>
<tr>
<td>-------------------------------</td>
</tr>
<tr>
<td>&nbsp;1 Liter</td>
</tr>
</tbody>
</table>
 </li>
<li>Filter the dissection medium using a 0.22 &micro;m filter. </li>
<li>Incubation medium (500ml) 
<table style="width: 283px; height: 157px;" border="0">
<tbody>
<tr>
<td>485 ml of filtered dissection media</td>
</tr>
<tr>
<td>5 ml of 1% N2 supplement (Gibco/Invitrogen)</td>
</tr>
<tr>
<td>10ml 1% horse serum (Sigma)</td>
</tr>
<tr>
<td>500 &micro;l of phenol red</td>
</tr>
<tr>
<td>----------------------------------------------------</td>
</tr>
<tr>
<td>&nbsp;0.5 L</td>
</tr>
</tbody>
</table>
 </li>
<li>Filter the incubation medium using a 0.22 &micro;m filter. </li>
<li>Bubble O2 into the dissection medium for 15 minutes before beginning the retina harvesting protocol. </li>
</ol>
Preparation of incubation chambers for retina
<ol>
<li>Under a hood, fill several 60 mm deep dishes (incubation chambers, Nunc) with 25 ml of incubation medium. The number of dishes depends on the number of retinas needed.</li>
<li>Place a filter stand into each deep dish. Only the very tips of the stand should not be submerged.</li>
<li>Place all deep dishes into the incubator until the retinas are ready.</li>
<li>Fill two 100 mm petri dishes and bring to the dissection bench.</li>
</ol>
Preparation of rabbit retina
<ol>
<li>Sacrifice a rabbit according to established protocols. The following does not have to be performed under a hood.</li>
<li>With angled scissors, insert it behind the eye into the eye socket of the rabbit, cutting the membranes and optical nerve that are attached to the eye.</li>
<li>Gently lift out the eye and wash it with oxygenated dissection media.</li>
<li>Place the eye (cornea facing up) into the well of an eye slicer and close the lid.</li>
<li>Place the blade horizontally right under the lid. In one smooth motion, draw the blade across the eye to cut off the cornea, leaving an eye cup.</li>
<li>Using forceps, remove the retina from the eye slicer well and place it onto a filter paper.</li>
<li>Remove the vitreous and lens by clamping an area near the severed optic nerve and pulling backwards on a filter paper. Repeat several times until almost all the vitreous is removed and the eye cup looks deflated.</li>
<li>Place eye cup into a petri dish with dissection media to wash.</li>
<li>Cut the eye cup into the desired number of pieces (5 pieces maximum).</li>
<li>Take one piece of eye cup and place it under the microscope.</li>
<li>Clamp the sclera and choroid lightly together on the edge of retina using forceps. With the other hand, use a fine brush to brush right between the choroid and retina.</li>
<li>Using short, gentle motions, tease the retina off the choroid until it becomes detached.</li>
<li>Cut 1 inch off a sterile transfer pipette. Use this to suck up the retina and place into a petri dish of incubation medium to wash.</li>
<li>Using another sterile transfer pipette with 1 inch cut off, carefully transfer the retina onto a 0.4 &micro;m Millicell filter (Millipore, Cat No: PICMORG50).</li>
<li>Use a brush to orient the retina with the ganglion cell side facing upwards.</li>
<li>Flatten out the retina by brushing lightly on the edges of the retina. Minimize touching the ganglion cell layer with the brush as much as possible.</li>
<li>Remove excess medium on filter with a transfer pipette.</li>
<li>Using forceps, transfer the filter onto a modified suctioner. Draw on the suctioner 2-3 times to remove all the incubation medium from the filter.</li>
</ol>
Gene gunning and incubation of retina
<ol>
<li>Under a hood, place each of the retina-containing-filters onto the filter stands in the 60 mm deep dishes (incubation chambers).</li>
<li>Ensure that the medium is in contact with the underside of the filter and that the medium does not spill over the sides of the filter onto the retina. The filter should act as an interface between the retina and medium.</li>
<li>Shoot each piece of retina with two bullets. Place gene gun directly above tissue, about 3-4 cm away.</li>
<li>Place all incubation chambers onto a shaker in the 35 - 37&deg;C incubator.</li>
<li>Replace with incubation medium every day. The retina can be incubated for a maximum of 6 days.</li>
</ol>

<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 ビュー.  MGH - Massachusetts General Hospital. この記事では、ウサギの網膜とGFPまたは網膜神経節細胞への細胞内マーカーの様々なコードするプラスミドの粒子による遺伝子導入の解剖とインキュベーションを示しています。

ビデオ: Organotypic Culture of Adult Rabbit Retina

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...