Name: isolation of the side population in myc-induced t-cell acute lymphoblastic leukemia in zebrafish
Uploaded: 2017-05-04T14:00:00
Description: Watch this Scientific Journal Video about Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish at JoVE.com

This work was supported by the University of Chicago Women's Board, the Wendy Will Case Fund, and the University of Chicago Comprehensive Cancer Center Support Grant (P30 CA014599). We would like to thank the Flow Cytometry Core at the University of Chicago for their help in setting up this experiment, and well as other members of the de Jong lab, particularly Leslie Pedraza and Sean McConnell.

All procedures with zebrafish have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Chicago. The University of Chicago is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
1. Generating and Isolating Fluorescently Labeled T-ALL Cells in Zebrafish
<ol>
	<li>Microinjection into syngeneic zebrafish embryos
	<ol>
		<li>Using commercially available kits, isolate circular rag2:c-myc and rag2...

<ol>
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USA. 101 (39), 14228-14233 (2004).</li><li>Komuro, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+side+population+cells+(stem-like+cell+population)+in+pediatric+solid+tumor+cell+lines.">Identification of side population cells (stem-like cell population) in pediatric solid tumor cell lines.</a> J Pediatr Surg. 42 (12), 2040-2045 (2007).</li><li>Moshaver, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+small+subpopulation+of+candidate+leukemia-initiating+cells+in+the+side+population+of+patients+with+acute+myeloid+leukemia.">Identification of a small subpopulation of candidate leukemia-initiating cells in the side population of patients with acute myeloid leukemia.</a> Stem Cells. 26 (12), 3059-3067 (2008).</li></ol>

The side population assay is highly sensitive; therefore, small changes to the protocol can result in a difficulty in detecting side population cells. First, the temperature during the staining step is specific to each animal/cell system. For mammalian systems, the side population assay is typically performed at 37 &#176;C2. When the zebrafish T-ALL cells were incubated at 37 &#176;C, many of the cells died, which made this incubation temperature unacceptable (data not shown). When Kobayashi and c...

The side population assay capitalizes on the enhanced ability of certain cells within a tissue to efflux the DNA binding dye Hoechst 33342 due to high levels of the ATP binding cassette (ABC) transporter proteins on the cell membrane. The cells that efflux the Hoechst 33342 dye can be identified using dual wavelength flow cytometric analysis after the dye is excited by a UV laser. This assay was first used to identify murine hematopoietic stem cells (HSCs)1, but it has since been used to identify stem/progenitor cell populations in many tissues and cancers (reviewed in reference 2). However, not all populations of cells have a side population, and not all side populations are enriched for stem/progenitor cells.
The zebrafish is a powerful vertebrate genetic model system for studying human cancer3,4, with a number of advantages over traditional murine models of cancer. Zebrafish embryos are externally fertilized and are optically clear, facilitating transgenesis and the in vivo observation of pathologic processes, including cancer initiation and progression. To date, the side population assay to detect potential stem or progenitor cells has only been applied to the kidney marrow in zebrafish to identify HSCs, and not to any zebrafish cancer models5,6.
The zebrafish model of T-cell acute lymphoblastic leukemia (T-ALL) is morphologically and genetically similar to human T-ALL7,8,11. T-ALL is an aggressive malignancy that, in humans, accounts for 10-15% of pediatric and 25% of adult ALL cases9. While the treatment of T-ALL has improved, relapse is still common and is associated with a poor prognosis. T-ALL tumors are heterogeneous and contain many different tumor cell subpopulations, including leukemia initiating cells (LICs). LICs are defined by their ability to regrow the entire tumor from a single cell, and the frequency of LICs within a tumor cell population can be calculated by transplanting varying cell doses into recipients via a limiting dilution transplantation assay (LDA). While LDA experiments have been performed in zebrafish to calculate the frequency of LICs8,10,11, this determination is made in hindsight and does not allow for the prospective isolation of LICs. Therefore, a method to prospectively isolate a population enriched for cancer stem cell activity is lacking. Identifying and isolating side population cells from zebrafish T-ALLs is the first step towards addressing this deficiency.
The protocol presented here describes how to efficiently generate T-ALL tumors in zebrafish utilizing tol2-mediated transgenesis, harvest the T-ALL tumors cells, and stain them with Hoechst 33342 to identify the side population of cells. Future in vivo experiments with zebrafish T-ALL could include whether the side population cells are enriched for LICs or have other stem- or progenitor-like properties.

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		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Syngeneic zebrafish (CG2)</td>
			<td style="width:199px;"></td>
			<td style="width:208px;"></td>
			<td style="width:208px;">See Mizgireuv et al., 2006</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">rag2:c-myc plasmid</td>
			<td style="width:199px;"></td>
			<td style="width:208px;"></td>
			<td>Langenau Laboratory</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:245px;">rag2:GFP plasmid constructed in de Jong Lab</td>
			<td style="width:199px;"></td>
			<td style="width:208px;"></td>
			<td style="width:208px;">rag2 promoter from Langenau Laboratory</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">pCS2-transposase plasmid</td>
			<td style="width:199px;"></td>
			<td style="width:208px;"></td>
			<td>Chien Laboratory</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">NotI -HF restriction enzyme</td>
			<td style="width:199px;">New England Bio-Labs Inc.</td>
			<td>R3189S</td>
			<td>500 units in 25 &#181;L</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:245px;">mMessage Machine SP6 Transcription Kit</td>
			<td style="width:199px;">Ambion</td>
			<td style="width:208px;">Thermo Fisher Scientific - AM1340</td>
			<td>25 reactions</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">QIAGEN Plasmid Midi Kit</td>
			<td style="width:199px;">Qiagen, Inc.</td>
			<td style="width:208px;">12643</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">SteREO Discovery V8 Microscope</td>
			<td style="width:199px;">Carl Zeiss Microscopy</td>
			<td>495015-0008-000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Digital microinjector</td>
			<td style="width:199px;">Tritech Research</td>
			<td>MINJ-D</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Brass straight-arm needle holder</td>
			<td style="width:199px;">Tritech Research</td>
			<td>MINJ-4</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Magnetic base and stand</td>
			<td style="width:199px;">Tritech Research</td>
			<td>MINJ-HBMB</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Micromanipulator</td>
			<td style="width:199px;">Narishige International</td>
			<td style="width:208px;">MN153</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Glass capillaries</td>
			<td style="width:199px;">Sutter Instrument Co.</td>
			<td style="width:208px;">B100-50-10</td>
			<td>10 cm without filament</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Flaming/Brown micropipette puller</td>
			<td style="width:199px;">Sutter Instrument Co.</td>
			<td style="width:208px;">P-97</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Microloader pipette tips</td>
			<td style="width:199px;">Eppendorf North America Inc.</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Phenol Red solution</td>
			<td style="width:199px;">Sigma Life Science</td>
			<td style="width:208px;">P0290</td>
			<td>0.5% concentration 100 mL</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Plastic Transfer pipettes</td>
			<td style="width:199px;">Fisherbrand</td>
			<td>137119D</td>
			<td>graduated 7.5 mL bulb</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tricaine methanesulfonate (MS-222)</td>
			<td style="width:199px;">Western Chemical, Inc.</td>
			<td style="width:208px;">Fisher Scientific - NC0342409</td>
			<td>100 g</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Fetal bovine serum (FBS)</td>
			<td style="width:199px;">HyClone Laboratories, Inc.</td>
			<td style="width:208px;">Fisher Scientific - SH3007103</td>
			<td>500 mL</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Phosphate-Buffered Saline (PBS)</td>
			<td style="width:199px;">Gibco by Life Technologies</td>
			<td style="width:208px;">1001-023</td>
			<td>pH 7.4 (1x) - 500 mL</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Heparin sodium salt</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:208px;">2106</td>
			<td>15 - 300 unit vials</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">40 um mesh filter</td>
			<td style="width:199px;">Falcon Corning Brand</td>
			<td style="width:208px;">352340</td>
			<td>Case of 50</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Trypan blue</td>
			<td style="width:199px;">Sigma Life Science</td>
			<td style="width:208px;">T8154</td>
			<td>20 mL - Solution (0.4%)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Hoechst 33342</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:208px;">H3570</td>
			<td>10 mL</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Verapamil</td>
			<td style="width:199px;">Sigma Life Science</td>
			<td style="width:208px;">V4629</td>
			<td>1 g</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:199px;">Sigma Life Science</td>
			<td style="width:208px;">D8418</td>
			<td>100 mL</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">Propidium Iodide</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:208px;">P3566</td>
			<td>10 mL</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">FACSAria Fusion cell sorter</td>
			<td style="width:199px;">BD Biosciences</td>
			<td style="width:208px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">BD FACSDiva 8.0.1</td>
			<td style="width:199px;">BD Biosciences</td>
			<td style="width:208px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:245px;">FlowJo v10.2</td>
			<td style="width:199px;">FlowJo, LLC</td>
			<td style="width:208px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of the side population in myc-induced t-cell acute lymphoblastic leukemia in zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

To efficiently generate fluorescent myc-induced T-ALL tumors in zebrafish, circular DNA constructs flanked by tol2 transposase sites can be co-injected with tol2 RNA. Previous studies from the injection of linearized DNA into zebrafish embryos report a 5% transgenesis rate8. With the protocol adjusted to include tol2-mediated transgenesis, transgenesis rates ranging from 10-44% can be observed (Table 1).
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Watch this Scientific Journal Video about Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish at JoVE.com

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

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