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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+foreign+genes+into+intact+maize+cells+with+high-velocity+microprojectiles.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+protocol+for+biolistic+transfection+of+brain+slices+and+dissociated+cultured+neurons+with+a+hand-held+gene+gun.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biolistic+transfection+of+neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">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.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1.6um gold particles (microcarrier)</td>
<td>0.25g</td>
<td>Bio-Rad</td>
<td>1652264</td>
<td>&nbsp;</td>
<tr>
<td>1M CaCl2</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>21115</td>
<td>&nbsp;</td>
<tr>
<td>100% EtOH</td>
<td>1pint</td>
<td>Gold shield</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cartridge Tubing</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>1652412</td>
<td>&nbsp;</td>
<tr>
<td>Scintillation vials</td>
<td>20ml, 500/case</td>
<td>VWR</td>
<td>66021-668</td>
<td>&nbsp;</td>
<tr>
<td>Dessication pellets</td>
<td>&#x201C;Dricap&#x201D; </td>
<td>MTI (800-445-9890)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Water bath sonicator</td>
<td>model 50T</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cartridge holder</td>
<td>pack of 5</td>
<td>Bio-Rad</td>
<td>1652426</td>
<td>&nbsp;</td>
<tr>
<td>Barrel liner</td>
<td>pack of 5</td>
<td>Bio-Rad</td>
<td>1652417</td>
<td>&nbsp;</td>
<tr>
<td>Diffuser screen</td>
<td>pack of 5</td>
<td>Bio-Rad</td>
<td>1652475</td>
<td>&nbsp;</td>
<tr>
<td>O-ring</td>
<td>75 Viton, Size 007, Qty, 100</td>
<td>McMaster-Carr</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Screen (mesh)</td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>144258</td>
<td>&nbsp;</td>
<tr>
<td>PVP </td>
<td>20mg/ml in EtOH</td>
<td>Bio-Rad</td>
<td>&nbsp;</td>
<td>Comes with tubing from biorad</td>
</tr>
<tr>
<td>Tubing prep station</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>1652418</td>
<td>&nbsp;</td>
<tr>
<td>Tubing cutter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>1652422</td>
<td>&nbsp;</td>
<tr>
<td>Helios Gene-Gun</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>1652411</td>
<td>&nbsp;</td>
<tr>
<td>Gene gun hose </td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>&nbsp;</td>
<td>Comes with Gene-Gun</td>
</tr>
<tr>
<td>Spermidine</td>
<td>5g</td>
<td>Sigma</td>
<td>s-0266</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			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).		</div>

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.  

Watch this Scientific Journal Video about Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture at JoVE.com

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

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 ...

preparation-gene-gun-bullets-biolistic-transfection-neurons-slice

Research

JoVE Journal

Biology

52.3K vues.  University of California, Davis. Nous décrivons une méthode pour la préparation de l'ADN des balles d'or revêtues et de démontrer l'utilisation de ces balles à biolistically transfecter des neurones en culture des coupes d'hippocampe.

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

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 ...

Gene-gun Transfection of Hippocampal Neurons

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, ...

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.