Funding has been provided by NIH grants 5T32CA09216-26 (for J.S.B.), and K01 AR055619-01A1 and 3 K01 AR055619-03S1 (for D.M.L.), as well as by the Alex's Lemonade Stand Foundation, the Harvard Stem Cell Institute and the American Cancer Society.

1. DNA microinjection of zebrafish embryos
<ol>
<li>Linearize rag2::cMyc and rag2::GFP11 DNA constructs by digesting 10&mu;g of each plasmid with NotI at 37&deg;C overnight.</li>
<li>Phenol:chloroform extract and precipitate the plasmids, and resuspend each in 20&mu;L H2O.</li> 
<li>Determine the concentration of the DNA by running a 1:1, 1:5 and 1:10 dilution of each digested plasmid on a 1% agarose gel with 20&mu;L, 10&mu;L and 5&mu;L of a high-range DNA ladder. This type of quantification is generally more accurate than a spectrometer reading.</li>
<li>Dilute the DNA to a final concentration of 60ng/&mu;L in 1M KCl+0.5X TE for injection into CG1-strain (or other syngeneic strain) zebrafish embryos using 30ng/&mu;L rag2::cMyc + 30ng/&mu;L rag2::GFP.</li>
<li>Injections should be performed as previously demonstrated12, 13. Briefly, male and female zebrafish are set up in mating chambers the night before injection. On the day of injection, DNA is loaded into the injection needle, the pressure is adjusted so that a bubble with a 50&mu;m diameter is expelled (30pg DNA), which is measured by a stage micrometer. Then, embryos are injected at the one-cell stage, with the DNA being injected directly into the cell, and not into the yolk. Survival of the injected CG1 embryos and larvae is generally poor, and only 5% of fish will successfully incorporate the transgene; thus, &gt;600 embryos should be injected to ensure that some fish will develop leukemia.</li> 
<li>Store the injected embryos at 28.5&deg;C, and remove the dead embryos after 24hr. Animals are then transferred to large tanks at 5 days of life and raised as previously described14.</li>
</ol>
2. Screening for primary T-ALL in zebrafish larvae
<ol>
	<li>Approximately 28 days after injection, anesthetize larval zebrafish by adding 200&mu;L of 4ng/m Tricane-S to 25mL of fish system water in a petri dish.</li>
	<li>Examine the larvae for development of fluorescently-labeled T-ALL by using an epifluorescence microscope at 20X magnification, using a 485/20 excitation wavelength and 530/25 emission filter to detect GFP fluorescence (Figure 1).</li> 
	<li>Separate tumor positive larvae from those that are negative. Negative larvae can be examined at a later time point, once tumors may have grown larger, to ensure that no T-ALL positive fish were missed.</li>
	<li>Monitor T-ALL positive fish at least once at week using the epifluorescence microscope to track tumor growth.</li>
</ol>
3. Isolation and purification of fluorescently-labeled primary leukemia cells
<ol>
<li>When GFP-positive leukemia cells have overtaken &gt;50% of the animal, sacrifice the fish by adding 1mL of 4ng/mL Tricane-S in a petri dish containing 9mL fish system water.</li>
<li>Place the fish in a new petri dish containing 1mL 0.9X PBS with 5% Fetal Bovine Serum to buffer the cells. Macerate the fish with a razor blade, pipet the mixture to dissociate large cell clumps, then pass the cells through a 40&mu;m mesh strainer into a 50mL tube. It is not necessary to dissociate all tissue clumps into single cells.</li> 
<li>Pipet 500&mu;L of the cells to a 4mL polystyrene tube. Add 1&mu;L 1mg/mL propidium iodide (PI), which will label dead cells. Vortex, then keep the cells on ice.</li>
<li>Sacrifice a wild-type CG1 fish, macerate and strain in the same way as above to collect normal cells, which serve as a carrier for the tumor cells during transplant.Add 4mL 5% FBS in 0.9X PBS to the 50mL tube for a total volume of 5mL.</li> 
<li>Count the normal cells, dilute to 3x105 cells/mL in 5% FBS in 0.9X PBS, then pipet 100&mu;L/well of a 96-well plate.</li>
<li>Using a Fluorescence Activated Cell Sorter (FACS), sort GFP positive, PI negative tumor cells (Figure 2A,B) into the 96-well plate containing blood cells at the following doses: 10 cells/well into 48 wells, 100 cells/well into 24 wells, 1000 cells/well into 12 wells, and 10,000 cells/well into 6 wells. Additionally, 10,000 cells should be sorted into a well without normal blood cells, and reanalyzed using FACS to assess the purity and viability of the sorted cells (Figure 2C).</li> 
</ol>
4. Limiting dilution transplantation into adult zebrafish
<ol>
<li>Pellet the cells by centrifuging the 96-well plate at 2000xg for 10 minutes.</li>
<li>Remove 95&mu;L of the supernatant, and resuspend the cells in the remaining 5&mu;L.</li> 
<li>Hold an adult (&gt;60 days old) syngeneic zebrafish ventral side up, and inject the cell suspension into the peritoneal cavity, using a 26G/2&#x201D; micro-syringe. Zebrafish do not have to be anesthetized before transplant. Typically, 45 fish are transplanted with 10 leukemia cells, 20 are transplanted with 100 cells, 7 are transplanted with 1,000 cells and 5 fish are transplanted with 10,000 T-ALL cells.</li> 
</ol>
5. Analysis to determine leukemia-initiating cell frequency
<ol>
	<li>Approximately 28 days after transplant, examine the transplanted fish with an epifluoresence microscope using a 485/20 excitation wavelength and 530/25 emission filter to detect GFP fluorescence. Record the number of positive fish per total number of fish injected.</li> 
	<li>Re-examine the fish every 14 days for up to 90 days and record the number of positive fish. When a tumor-positive fish become moribund, sacrifice the animal by Tricane-S overdose.</li> 
	<li>Input the results into a table, as shown in Figure 3D. Upload the data into the web-based ELDA (Extreme Limiting Dilution Analysis) statistical software at <a href="http://bioinf.wehi.edu.au/software/elda/index.html">http://bioinf.wehi.edu.au/software/elda/index.html</a>15, which uses the frequency of tumor positive and negative animals at each transplant dose to determine the frequency of self-renewing leukemia cells within the primary T-ALL.</li>
</ol>
6. Representative Results:
DNA microinjection into embryos from syngeneic fish often results in a low survival rate of the injected embryos, with &lt;60% of embryos surviving for 24 hours. Additionally, survival of the injected syngeneic fish is generally poor during larval development, with often &lt;20% surviving for 28 days. It is therefore important to inject a large number of embryos in order to create transgenic fish that will develop T-ALL.
As an example, CG1 strain embryos were injected with rag2::cMyc+rag2::GFP. The transgenes co-segregate in the developing embryo, so that every cell that expresses GFP will also express cMyc. Larvae were screened for primary T-ALL after 28 days (Figure 1). Previous work has shown that approximately 5% of injected fish will have GFP-positive T-cells within their thymus (Figure 1), and 100% of these fish will develop leukemia as the T-cells become transformed11. While a small proportion of zebrafish may have a more advanced leukemia that is very easy to detect at day 28, most will only have a GFP-positive thymus (Figure 1), so larvae should be examined individually under high magnification to ensure that all positive fish are selected. GFP-positive T-ALL cells will overtake &gt;50% of the animal between days 45-70. 
In the example provided, zebrafish were sacrificed at 65 days of life, and leukemia cells were isolated and FACS sorted for limiting dilution transplantation. Single cells that were propidium iodide negative and GFP-positive (Figure 2) were sorted into a 96-well plate. Reanalysis was done on 10,000 sorted cells in order to assess viability (ideally &gt;95%) and purity (ideally &gt;92%, Figure 2). Transplanted T-ALLs generally arise before 28 days, but some tumors may take more than 60 days to develop. In this example, at day 28, early stage tumors were seen as an area of GFP-positive cells near the injection site (Figure 3B), while some T-ALLs were more progressed, having expanded to fill the peritoneal cavity (Figure 3C). Data from limiting dilution cell transplantation assays from three different primary T-ALLs are shown in Figure 3D. For these experiments, over 220 animals were transplanted, which can be easily accomplished by one person within several hours. Data analysis using ELDA software showed that, on average, 1 in 135 T-ALL cells were self-renewing leukemia-propagating cells (Figure 3E).
<img src="/files/ftp_upload/2790/2790fig1.jpg" alt="Figure 1"/> 
Figure 1. Zebrafish larvae injected at the one-cell stage with rag::-cMyc+rag::-eGFP were screened for primary leukemia growth 28 days post-injection. Fish (*) had GFP-positive T-cells within the thymus; this fish will develop T-ALL as the T-cells become transformed over time. This stage is very common at day 28. One fish (**) had an advanced T-ALL that has spread into the soft tissue. The remaining two fish are negative for tumor growth. The image was taken at 16X magnification.
<img src="/files/ftp_upload/2790/2790fig2.jpg" alt="Figure 2"/> 
Figure 2. Fluorescently-labeled T-ALL cells were sorted from diseased animals and used in the limiting dilution cell transplantation assay. First, a gate was drawn to select single cells (upper left panel), then propidium iodide negative cells are selected (middle left panel). Finally, a gate was drawn to select only the GFP-positive leukemia cells for sorting (lower left panel). Sorted cells should be reanalyzed to assess viability and purity before transplant (right panels).
<img src="/files/ftp_upload/2790/2790fig3.jpg" alt="Figure 3"/> 
Figure 3. Zebrafish were examined for leukemia growth 28 days after transplant. Fish are either tumor negative (A), have a small tumor growing at the injection site (B), or have a progressed leukemia (C). The images were taken at 7X magnification. The total number of leukemia-positive fish per total number of fish transplanted at each dilution is recorded, as in (D). The data are input into the web-based ELDA program to calculate the number of self-renewing leukemia cells and the upper and lower 95% confidence intervals (E).

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Tumor+Assays:+The+State+of+Transplantation.">Zebrafish Tumor Assays: The State of Transplantation.</a> Zebrafish. 6, 339-346 (2009).</li><li>Mizgirev, I., Revskoy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+clonal+zebrafish+lines+and+transplantable+hepatic+tumors.">Generation of clonal zebrafish lines and transplantable hepatic tumors.</a> Nat Protocols. 5, 383-394 (2010).</li><li>Mizgirev, I. V., Revskoy, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantable+Tumor+Lines+Generated+in+Clonal+Zebrafish.">Transplantable Tumor Lines Generated in Clonal Zebrafish.</a> Cancer Research. 66, 3120-3125 (2006).</li><li>Smith, A. 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No conflicts of interest declared.

A major strength of using zebrafish in cancer research is that large numbers of animals can be used at relatively low cost. This is especially important in limiting dilution cell transplantation assays, where the proportions of transplanted animals that develop tumors to the total number transplanted are used to determine tumor-initiating cell frequency. In the method presented here, over 70 transplant recipient animals are used per assay, providing an accurate estimate of tumor-propagating cell number. Both the number of animals used and the doses of tumor cells transplanted should be optimized for a given cancer model; for example, if initial experiments show tumor-initiating cells are rare, useful transplant doses may be 100,000, 50,000, 10,000 and 1,000 cells per transplant.
Syngeneic zebrafish strains are useful in limiting dilution analysis. However, these strains are not yet commonly used in all laboratories, and some zebrafish cancer models exist only in allogenic fish. Limiting dilution cell transplantations can be performed in wild-type strains that have undergone immune ablation, although the self-renewing cell frequency may be calculated as 10-100 fold higher than if syngeneic zebrafish were used8. It is important to note that greater doses of cells should be used for transplants into non-immune matched, irradiated recipients, as some tumor cells will not survive transplant into a foreign microenvironment. Additionally, many tumors will begin to regress after 21 days when the recipient animal's immune system recovers, so animals should be assessed for tumor growth every 7 days after transplant.
The methods presented here are suitable for use with any zebrafish cancer model, and have thus far been used to determine tumor-initiating cell frequency in both T-cell acute lymphoblastic leukemia8, and a solid tumor model, rhabdomyosarcoma16. These tumors were fluorescently labeled by co-injection of embryos with both an oncogene and fluorescent marker; because the DNA co-segregates, any cell expressing the oncogene will also express the fluorescent protein17. While fluorescence is recommended because it facilitates tracking of tumor growth without the need to sacrifice the animal and provides a straight-forward way to isolate tumor cells by FACS, it is possible to label tumor cells with antibodies to tumor-specific makers to facilitate their purification, though antibody staining in zebrafish may require optimization.
Finally, once the incidence of tumor-propagating cells has been determined for a given tumor type, it is possible to use these assays to identify the mechanisms that govern their cell frequency. For example, primary tumor cells can be treated with pathway specific inhibitors before limiting dilution transplant, or transplanted fish themselves may be dosed with drugs. Additionally, the genetics of the tumors themselves may be manipulated by injecting the one-cell stage fish with a gene of interest, so that the resulting primary tumors will over-express the gene. In these experiments, any effect on self-renewing cell frequency will be observed in the limiting dilution assays.

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the Reagent</th>
 <th>Company</th>
 <th>Catalog Number</th>
 </tr> 	
 <tr>
 	<td>NotI restriction enzyme</td>
 <td>New England Biolabs</td>
 <td>R0189</td>
 </tr>
 <tr>
 	<td>Phenol:Chloroform</td>
 <td>EMD Biochemicals</td>
 <td>6805-OP</td>
 </tr>
 <tr>
 	<td>5M KCl</td>
 <td>Sigma-Aldrich</td>
 <td>P9541</td>
 </tr>
 <tr>
 	<td>100X Tris-EDTA</td>
 <td>Sigma-Aldrich</td>
 <td>T9285</td>
 </tr>
 <tr>
 	<td>High Range DNA ladder</td>
 <td>Fermentas</td>
 <td>R0621</td>
 </tr>
 <tr>
 	<td>Syngeneic zebrafish</td>
 <td>&nbsp;</td>
 <td> References6-8</td>
 </tr>
 <tr>
 	<td>Fetal Bovine Serum</td>
 <td>Gibco</td>
 <td>16000-036</td>
 </tr>
 <tr>
 	<td>Propidium Iodide</td>
 <td>Sigma-Aldrich </td>
 <td>P4864 </td>
 </tr>
 <tr>
 	<td>26G/2" Microsyringe</td>
 <td>Hamilton</td>
 <td>80366</td>
 </tr>
 <tr>
 	<td>40&mu;m mesh cell strainer</td>
 <td>BD Falcon</td>
 <td>352340</td>
 </tr>
 </tbody>
</table>

quantifying the frequency of tumor-propagating cells using limiting dilution cell transplantation in syngeneic zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Watch this Scientific Journal Video about Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish at JoVE.com

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

quantifying-frequency-tumor-propagating-cells-using-limiting-dilution

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19.1K ビュー.  Harvard Medical School. 限界希釈細胞移植アッセイは、腫瘍増殖する細胞の頻度を決定するために使用されます。このプロトコルは、蛍光標識された白血病と成人のゼブラフィッシュの腹腔内に限界希釈法でこれらの白血病細胞を分離して移植する方法の詳細を開発する同系のゼブラフィッシュを生成するための方法を説明します。

ビデオ: Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell therapy may alleviate many of the symptoms associated with lung disease and induce alveolar repair in adults. Concurrent with the ongoing search for stem cells applicable for human treatment, precise delivery and homing (to the site of disease) must be reassured for successful therapy. Here, I report that stem cells can safely be instilled via the trachea opening a non-stop route to the lung. This method involves a skin incision, caudal insertion of a cannula into and along the tracheal lumen, and injection of a stem cell vehicle mixture into airways of the lung. A broad range of media solutions and stabilizers can be instilled via tracheotomy, resulting in the ability to deliver a wider range of cell types. With alveolar epithelium confining these cells to the lumen, lung expansion and negative pressure during inhalation may also assist in stem cell integration. Tracheal delivery of stem cells, with a quick uptake and the ability to handle a large range of treatments, could accelerate the development of cell-based therapies, opening new avenues for treatment of lung disease.

Significant breakthroughs in stem cell identification are continuously being made. To translate these discoveries, however, novel methods for cellular delivery must be devised. Here I report that the airways provide a safe route for stem cell transplantation to the lungs.

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

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 ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...