This work is supported by the V Foundation Scholar (AGF) and the MPN Research Foundation (AGF).

This study was approved and carried out in accordance with the recommendations by the Institutional Animal Care and Use Committee at University of California, Irvine. All procedures were performed under isoflurane anesthesia and all efforts were made to minimize suffering.
1. Generation of Ecotropic Retrovirus
<ol>
	<li>Prepare high-quality plasmids with a concentration of at least 1 &#181;g/&#181;l using either a commercial maxi-prep kit or cesium chloride purification. 
	NOTE: These inclu...

<ol>
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S., 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=Molecular+pathogenesis+and+therapy+of+polycythemia+induced+in+mice+by+JAK2+V617F.">Molecular pathogenesis and therapy of polycythemia induced in mice by JAK2 V617F.</a> PloS One. 1, e18 (2006).</li><li>Bumm, T. G. P., Elsea, C., 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=Characterization+of+murine+JAK2V617F-positive+myeloproliferative+disease.">Characterization of murine JAK2V617F-positive myeloproliferative disease.</a> Cancer Res. 66 (23), 11156-11165 (2006).</li><li>Marty, C., Pecquet, C., 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=.">.</a> Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis. , (2015).</li><li>Klampfl, T., Gisslinger, 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=Somatic+mutations+of+calreticulin+in+myeloproliferative+neoplasms.">Somatic mutations of calreticulin in myeloproliferative neoplasms.</a> N. Engl. J. Med. 369 (25), 2379-2390 (2013).</li><li>Araki, M., Yang, Y., 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=Activation+of+the+thrombopoietin+receptor+by+mutant+calreticulin+in+CALR-mutant+myeloproliferative+neoplasms.">Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms.</a> Blood. , (2016).</li><li>Lim&#243;n, A., Briones, J., 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=High-Titer+Retroviral+Vectors+Containing+the+Enhanced+Green+Fluorescent+Protein+Gene+for+Efficient+Expression+in+Hematopoietic+Cells.">High-Titer Retroviral Vectors Containing the Enhanced Green Fluorescent Protein Gene for Efficient Expression in Hematopoietic Cells.</a> Blood. 90 (9), 3316-3321 (1997).</li><li>Duran-Struuck, R., Dysko, R. C. <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+bone+marrow+transplantation+(BMT):+providing+optimal+veterinary+and+husbandry+care+to+irradiated+mice+in+BMT+studies.">Principles of bone marrow transplantation (BMT): providing optimal veterinary and husbandry care to irradiated mice in BMT studies.</a> J. Am. Assoc. Lab. Anim. Sci. 48 (1), 11-22 (2009).</li><li>Gavrilescu, L. C., Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Replication-Defective+Retrovirus+by+Transient+Transfection+of+293T+cells.">Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells.</a> J. Vis. Exp. (10), (2007).</li><li>Challen, G. A., Boles, N., Lin, K. K., Goodell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Hematopoietic+Stem+Cell+Identification+And+Analysis.">Mouse Hematopoietic Stem Cell Identification And Analysis.</a> Cytometry A. 75 (1), 14-24 (2009).</li><li>Ergen, A. V., Jeong, M., Lin, K. K., Challen, G. A., Goodell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+mouse+side+population+cells.">Isolation and characterization of mouse side population cells.</a> Methods Mol. Bio. (Clifton, N.J.). 946, 151-162 (2013).</li><li>Weksberg, D. C., Chambers, S. M., Boles, N. C., Goodell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD150-+side+population+cells+represent+a+functionally+distinct+population+of+long-term+hematopoietic+stem+cells.">CD150- side population cells represent a functionally distinct population of long-term hematopoietic stem cells.</a> Blood. 111 (4), 2444-2451 (2008).</li><li>Oguro, H., Ding, L., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+markers+resolve+functionally+distinct+subpopulations+of+hematopoietic+stem+cells+and+multipotent+progenitors.">SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.</a> Cell Stem Cell. 13 (1), 102-116 (2013).</li><li>Li, J., Kent, D. G., Chen, E., Green, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+myeloproliferative+neoplasms:+JAK+of+all+grades.">Mouse models of myeloproliferative neoplasms: JAK of all grades.</a> Dis. Model. Mech. 4 (3), 311-317 (2011).</li><li>Mullally, A., Lane, S. W., Brumme, K., Ebert, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloproliferative+neoplasm+animal+models.">Myeloproliferative neoplasm animal models.</a> Hematol. Oncol. Clin. North Am. 26 (5), 1065-1081 (2012).</li></ol>

This protocol provides a detailed description of how to perform bone marrow transplants in mice to recapitulate an essential thrombocythemia-like disease with progression to myelofibrosis with CALRdel52 mutation as the driver of disease. Successful transplantation of cells expressing CALRdel52 results in increased platelets, expansion of megakaryocytes, and bone marrow fibrosis. As bone marrow transplantation is a multistep process, it is important to acknowledge steps where technical error can be avoided to prevent poor...

Transduction-transplantation is a useful method to model hematologic malignancies in mice. This technique has been particularly valuable for studying myeloid malignancies dating back to the first demonstration that ectopic expression of BCR-ABL1 could faithfully recapitulate chronic myelogenous leukemia in mice1. This technique has subsequently facilitated the extensive study of JAK2V617F and MPLW515K/L mutated myeloproliferative neoplasm (MPN).
MPN are a group of hematologic malignancies characterized by the overproduction of mature myeloid cells and bone marrow fibrosis. These diseases generally arise from the clonal expansion of a hematopoietic stem cell that has acquired a somatic mutation in either Jak2, MPL, or CALR. Transduction-transplantation JAK2V617F and MPLW515K/L models exhibit the clinical features of polycythemia vera and myelofibrosis2-5. Recently, a mouse model of calreticulin-mutated MPN has also been generated with the transduction-transplantation method6. These mice develop an essential thrombocythemia-like disease with increased platelets, increased number of megakaryocytes, and bone marrow fibrosis. Together, these models have not only provided the opportunity to gain insight into the molecular pathogenesis of MPN, but also the capacity to develop and study therapeutics in a pre-clinical setting.
This manuscript provides a detailed description of transduction-transplantation methodology with a focus on the CALRdel52 mutation. This technique involves transplantation of retrovirally transduced bone marrow cells expressing the mutant construct into irradiated syngeneic recipient mice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CELL LINES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>MT-10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>3T3 cells</td>
			<td>ATCC</td>
			<td>CRL-1658</td>
			<td></td>
		</tr>
		<tr>
			<td>PLASMIDS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EcoPak, also known as pCL-Eco</td>
			<td>Addgene</td>
			<td>12371</td>
			<td>Retroviral packaging cell lines, such as EcoPack 2-293, may be used in place of the EcoPak plasmid and standard 293T cells. Additional &#947;-retrovirus envelope and packaging plasmids are available from Addgene and others.</td>
		</tr>
		<tr>
			<td>MSCV-IRES-GFP (MIG)</td>
			<td>Addgene</td>
			<td>20672</td>
			<td>Additional &#947;-retroviral transfer plasmids are available from Addgene and others.</td>
		</tr>
		<tr>
			<td>CONSUMABLES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>27G x 1/2&#34; needles</td>
			<td>BD</td>
			<td>305620</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Corning</td>
			<td>MT-35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin/L-glutamine</td>
			<td>Corning</td>
			<td>MT-30-009-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>Corning</td>
			<td>MT-25-052-CI</td>
			<td>Can be homemade</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>MT-21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>10cm dishes</td>
			<td>Fisher</td>
			<td>172931</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Fisher</td>
			<td>12565268</td>
			<td></td>
		</tr>
		<tr>
			<td>60mm dishes</td>
			<td>Fisher</td>
			<td>150288</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Fisher</td>
			<td>NC9840454</td>
			<td></td>
		</tr>
		<tr>
			<td>5-FU</td>
			<td>Fisher</td>
			<td>A13456-06</td>
			<td></td>
		</tr>
		<tr>
			<td>100um cell strainers</td>
			<td>Fisher</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Fisher</td>
			<td>12565270</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Fisher</td>
			<td>130184</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Fisher</td>
			<td>14-959-5</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45um syringe filters</td>
			<td>Fisher</td>
			<td>0974061B</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK buffer</td>
			<td>Lonza</td>
			<td>10-548E</td>
			<td>Can be homemade</td>
		</tr>
		<tr>
			<td>Recombinant murine IL-3</td>
			<td>Peprotech</td>
			<td>213-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine IL-6</td>
			<td>Peprotech</td>
			<td>216-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine SCF</td>
			<td>Peprotech</td>
			<td>250-03</td>
			<td></td>
		</tr>
		<tr>
			<td>X-tremeGENE 9</td>
			<td>Roche</td>
			<td>6365809001</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>1.5 ml centrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Accuri C6</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray irradiator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

transduction-transplantation mouse model of myeloproliferative neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

The transduction-transplantation technique results in hematopoietic reconstitution of the recipient mice with cells expressing the gene of interest. Figure 1 shows an overview of the transduction-transplantation mouse model of calreticulin mutated MPN. Briefly, retrovirus expressing CALRwt or CALRdel52 is used to infect BM cells from a C57B/6 donor mouse. Transduced cells are transplanted into irradiated C57B/6 recipient mouse and donor cell engraftment occurs during the ...

Watch this Scientific Journal Video about Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm at JoVE.com

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

The overall goal of this procedure is to generate mice expressing the gene of interest in hematopoietic cells using the transduction-transplantation method. Within a short period of time, this method can help answer whether your gene of interest induces a hematologic malignancy. The implications of this technique extend towards therapy, or diagnoses of hematological malignancies, because of the expedited development of mouse models that can test putative therapeutics.Although this method can provide insight into hematological malignancy, it can also be used to study the role of a gene of interest of normal hematopoiesis. Generally, individuals new to this experiment will struggle, as it requires multiple steps. Also, generation of a high titer virus is critical to the success of this experiment.Visual demonstration of this procedure is important, as working with mice can be difficult, and requires practice. After anesthetizing a mouse, and checking for depth of anesthesia according to the text protocol, position the mouse on its side, and using the thumb and index finger, restrain the animal so that the eye protrudes slightly from the head. To inject 150 milligrams per kilogram of 5-flurouracil, or 5-FU, via a retro-orbital injection, insert a 27-gauge half-inch needle at a 45-degree angle with the bevel facing up in a medial direction, and stop when the needle is halfway inserted.Slowly inject the solution, and then carefully remove the needle. Examine the mouse for any signs of injury, such as swelling or bleeding, and ensure that the mouse recovers and begins to walk. Five days later, after sacrificing the animal according to the text protocol, use 70%ethanol to sterilize the mouse by spraying generously until the fur becomes wet.With dissecting scissors, make an incision in the skin, and dissect the fascia from the leg muscles. Then, dissect the leg away from the mouse by severing the femur at the hip, and the tibia at the ankle. Bend the leg into an L-shape and use dissecting scissors to separate each leg bone by cutting at the knee joint.Then, gently scrape the muscle towards one end of the leg bone until the cartilage is disengaged. Next, using a syringe with a 27-gauge half-inch needle, in PBS supplemented with 2%FBS, flush each leg bone over a 100 micrometer nylon membrane into a 50 milliliter conical tube, until the bone turns white to maximize the number of cells harvested. With the plunger of a syringe, mash the cells through the nylon membrane into the 50 milliliter conical tube to obtain a single cell suspension.Repeat with the other mouse leg. Centrifuge the harvested cells at 400 times g in four degrees Celsius for 10 minutes, and discard the supernatant. Then, at five milliliters of ammonium chloride potassium, or ACK, to re-suspend the bone marrow.Incubate the cell suspension on ice for 10 minutes to lyse the red blood cells. To stop the lysis, use PBS to fill the tube. Then, centrifuge the cells again, and discard the supernatant.With five milliliters of PBS, re-suspend the cells, and by Trypan blue exclusion, use a hemocytometer to count the cells. Now, add 2x pre-stem cocktail to re-suspend the cells at two times 10 to the sixth cells per milliliter. Then, plate two milliliters in the wells of six-well plates.Incubate the cells at 37 degrees Celsius, and 5%carbon dioxide overnight. After the incubation, add two milliliters of viral supernatant to each well, followed by eight micrograms per milliliter of polybrene. Spin the plates at 1500 times g in 30 degrees Celsius for 90 minutes.Then return the plates to the incubator overnight. Pipette the cell suspension into conical tubes, and centrifuge at 400 times g in four degrees Celsius for 10 minutes. Use 2x pre-stem cocktail per well to re-suspend the cells, and re-plate in the original wells.After preparing recipient mice for transplantation according to the text protocol, harvest transduced donor cells from the six-well plates by gently pipetting supernatant into conical tubes. Use one milliliter of PBS to wash each well, and collect the residual cells in the tubes. Add 0.5 milliliters of 0.05%trypsin to the wells, and incubate at 37 degrees Celsius for five minutes.Next, add one milliliter of D-10 to the wells, and detach any remaining cells by gently pipetting. Then, add the cells to the respective tubes and pellet at 400 times g in four degrees Celsius for 10 minutes. With PBS, wash the cells again.Then, after spinning, use five milliliters of PBS to re-suspend the cells, and count them using Trypan blue exclusion and a hemocytometer. For injection into mice, re-suspend five times 10 to the fifth, through two times 10 to the sixth cells in 50 to 100 microliters of PBS. Position the mouse on its side, and restrain it by using the thumb and index finger, so that the eye protrudes slightly from the head.With the bevel facing up, insert the needle lateral to the medial canthus at a 45-degree angle, and inject. Following the injection, examine the mouse for any signs of injury, such a swelling or bleeding, at the injection site, and refer to the text protocol for additional post-injection care. Finally, analyze GFP expression in blood, according to the text protocol.This figure illustrates how to calculate relative viral titer where the percent GFP is calculated from events collected from the live cell gate. Shown here is a titer with more than one viral particle per cell, and these samples represent titers with single viral particles per cell. Ideally, at least 50%of GFP-positive cells would be detected with a one to 30 dilution of viral supernatant.In this experiment, a successful transplant recipient is presented where CALRdel52 mice exhibit an MPN phenotype with increased platelets, increased megakaryocytes in the bone marrow, and bone marrow fibrosis. Once mastered, this technique can be done in eight days, if performed properly. While attempting this procedure, it is important to follow every step of the protocol.After its development, this technique paved the way for researcher to explore the effects of specific genes in hematopoiesis. After watching this video, you should have a good understanding of how to produce mice expressing your gene of interest in hematopoietic cells.

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11.7K 次观看.  University of California, Irvine. The overall goal of this procedure is to generate mice expressing the gene of interest in hematopoietic cells using the transduction-transplantation method. Within a short period of time, this method can help answer whether your gene of interest induces a hematologic malignancy. The implications of this technique extend towards therapy, or diagnoses of hematological malignancies, because of the expedited development of mouse models that can test putative therapeutics.Although this meth...