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 include an MSCV backbone vector encoding the gene of interest (in this case CALR7,8) and marker of choice (GFP, Neo, etc.) as well as an ecotropic packaging plasmid, encoding gag-pol-env (referred here as plasmid).</li>
	<li>Thaw and expand low-passage 293T cells in DMEM supplemented with 10% FBS and L-glutamine with penicillin/streptomycin (D10). Plate 5 x 106 cells in a 10 cm tissue culture-treated dish in a humidified tissue culture incubator at 37 &#176;C and 5% CO2.</li>
	<li>Expand 293T cells in culture. On the day before transfection, wash cells by adding 1 ml PBS and aspirate supernatant into a vacuum flask.</li>
	<li>To trypsinize cells, add 2 ml 0.05% trypsin and incubate dishes in a humidified tissue culture incubator for 2 - 3 min at 37 &#176;C and 5% CO2.</li>
	<li>To stop trypsinization, add 3 ml D10 to dish and transfer cells to a 15 ml conical tube. Spin cells at 400 x g for 10 min.</li>
	<li>Aspirate supernatant into a vacuum flask and resuspend cell pellet in 2 ml D10.</li>
	<li>Count the cells by trypan blue exclusion using a hemocytometer.</li>
	<li>Seed 2 x 106 cells per dish into three 10 cm tissue culture-treated dishes per virus generated. 
	NOTE: For optimal results, do not allow the cells to exceed 30 - 50% confluence at time of transfection or a poor virus yield may be obtained.</li>
	<li>Immediately before transfection, aspirate media into a vacuum flask and replace with 5 ml fresh D10.</li>
	<li>For every 10 cm Petri dish to be transfected prepare an individual sterile 1.5 ml microcentrifuge tube.</li>
	<li>Pipette 760 &#181;l of reduced serum media into each microcentrifuge tube, then carefully add 40 &#181;l of transfection reagent directly into the media. Incubate tubes in the tissue culture hood at room temperature for 5 min.</li>
	<li>Add 30 &#181;g of MSCV vector with gene of interest (15 &#181;g if using empty vector) as well as 5 &#181;g of plasmid to each respective tube and mix gently by pipetting up and down. Incubate at room temperature in the tissue culture hood for an additional 15 min. 
	NOTE: Smaller vectors tend to have higher viral yields.</li>
	<li>Carefully add the total volume of each reaction (approximately 800 &#181;l) drop-wise into its respective 10 cm Petri dish and gently tilt the dish from side to side to mix. Return cells to the humidified tissue culture incubator.</li>
	<li>Following an 8 hr or overnight incubation in tissue culture incubator, aspirate media into a vacuum flask and gently replace with 10 ml of fresh D10.</li>
	<li>At 48 hr post-transfection collect supernatant into a 35 ml syringe and filter through a 0.45 &#181;m membrane to remove cell debris.</li>
	<li>OPTIONAL: Place 10 ml fresh D10 back on the 293T cells and additional virus may be harvested at 72 hr post-transfection.</li>
	<li>Dispose of 293T cells in biohazard waste when no additional virus is collected.</li>
	<li>Aliquot viral supernatant into single use volumes (for example 1 ml for virus titer or 6.5 ml for BM infection). Flash-freeze virus with liquid nitrogen and store virus at -80 &#176;C. 
	NOTE: Avoid repeated freeze/thaw cycles as they greatly reduce virus titer.</li>
</ol>
2. Determine Relative Viral Titer by Flow Cytometry
<ol>
	<li>Thaw 3T3 cells and maintain in D10. Culture the cells in a 10 cm tissue-culture treated dish in a humidified tissue culture incubator at 37 &#176;C and 5% CO2.</li>
	<li>Perform viral titer in triplicate. To prepare cells, wash cells by adding 1 ml PBS and aspirate supernatant into a vacuum flask.</li>
	<li>To trypsinize cells, add 2 ml 0.05% trypsin and incubate dishes in a humidified tissue culture incubator at 37 &#176;C and 5% CO2.</li>
	<li>To stop trypsinization, add 3 ml D10 to dish and transfer cells to a 15 ml conical tube. Spin cells at 400 x g for 10 min.</li>
	<li>Aspirate supernatant into a vacuum flask and resuspend cell pellet in 2 ml D10.</li>
	<li>Count the cells by trypan blue exclusion using a hemocytometer.</li>
	<li>Seed 3T3 cells at a density of 1 x 105 cells into fifteen 60 mm tissue culture-treated dishes and incubate overnight.</li>
	<li>Make 1:3, 1:10, 1:30, and 1:100 dilutions of thawed viral supernatant using D10 in a total of 1 ml. Also include a virus-free control.</li>
	<li>Aspirate media from 3T3 cells into a vacuum flask and replace with 4 ml of D10.</li>
	<li>Overlay the 3T3 cells with the dilutions of viral supernatant and add polybrene to a final concentration of 8 &#181;g/ml to each dish. Incubate for 48 hr in the tissue culture incubator at 37 &#176;C and 5% CO2.</li>
	<li>Determine relative viral titer by flow cytometry:
	<ol>
		<li>To wash cells, aspirate the media into a vacuum flask and add 5 ml PBS.</li>
		<li>Aspirate the PBS into a vacuum flask. Add 1 ml 0.05% trypsin and incubate at 37 &#176;C for 2 - 5 min to detach cells from dish.</li>
		<li>Add 3 ml D10 and pipette up and down to detach any remaining cells from the plate. Transfer the cells to a FACS tube and centrifuge at 400 x g for 10 min.</li>
		<li>Decant supernatant and wash cells with 3 ml PBS. Centrifuge at 400 x g for 10 min and decant supernatant. Resuspend in 200 &#181;l PBS.</li>
		<li>Run cells on a flow cytometer9. Collect between 20,000 - 30,000 events within the live cell gate (determined by FSC vs. SSC) and calculate the percentage of GFP+ and GFP- cells. 
		NOTE: The viral titer is considered acceptable when the 1:30 dilution of viral supernatant yields at least 50% GFP+ 3T3 cells. If this dilution results in less than 50% GFP+ cells then suboptimal transduction results may be obtained. The calculation to determine viral titer was adapted from Limon et al., Blood (1997)9.</li>
		<li>To determine relative virus titer, use the equation: virus titer = particle forming units (PFU)/ml = [(Number of 3T3 Cells &#215; Frequency of GFP+ Cells) / Volume of Supernatant (ml)] &#215; Dilution Factor.</li>
	</ol>
	</li>
</ol>
3. Transduce Donor Bone Marrow Cells
<ol>
	<li>To prepare donor mice for bone marrow (BM) harvest, first anesthetize mice by isoflurane vaporizer with a flow rate of 5% combined supplemental oxygen at 1 L/min. Pinch foot to assess responsiveness. 
	NOTE: Donor mice (less than 8 weeks old) should be prepared 5 days prior to bone marrow harvest (8 days prior to transplantation). One donor mice will provide enough cells for at least 2 recipients.</li>
	<li>Inject 150 mg/kg 5-fluorouracil (5-FU) via retro-orbital injection in anesthetized mice using a 27 G &#215; &#189;&#34; needle. 
	CAUTION! 5-FU an anti-cancer chemotherapeutic agent. Always handle 5-FU inside a certified chemical fume hood or a ducted biosafety cabinet. Consult with the institution&#39;s laboratory and animal safety committee to determine appropriate labeling of mice treated with 5-FU.
	<ol>
		<li>Position the anesthetized mouse on its side and restrain by using both the thumb and index finger such that the eye protrudes slightly from the head.</li>
		<li>Starting with the needle lateral to the medial canthus and with the bevel facing up, insert the needle at a 45&#176; angle in the medial direction and stop when the needle is halfway inserted. Slowly inject cells and remove carefully remove needle. 
		NOTE: If this technique is performed correctly, the eye should retract slightly and no resistance should be felt upon needle insertion.</li>
		<li>Examine mouse for any signs of injury such as swelling or bleeding.</li>
	</ol>
	</li>
	<li>Harvest bone marrow 5 days after 5-FU treatment (3 days prior to transplantation).
	<ol>
		<li>Anesthetize mice by isoflurane vaporizer with a flow rate of 5% combined supplemental oxygen at 1 L/min. Pinch foot to assess responsiveness.</li>
		<li>Sacrifice mouse by cervical dislocation. Sterilize mouse by spraying generously with 70% EtOH until fur becomes wet.</li>
		<li>Using dissecting scissors, make an incision in the skin and dissect the fascia away from the leg muscles. Next, dissect the leg away from mouse by severing the femur at the hip and the tibia at the ankle.</li>
		<li>Bend the leg into a &#34;L&#34; shape and separate each leg bone by cutting at the knee joint with dissecting scissors.</li>
		<li>To remove cartilage at the distal ends of each leg bone, use the dissecting scissors to gently scrape muscle towards one end of the leg bone until cartilage is disengaged.</li>
		<li>Using a syringe with a 27 G x &#189; inch needle, flush each leg bone with PBS supplemented with 2% FBS over a 100 &#181;M nylon membrane in a 50 ml conical tube. Flush bone until bone turns white to maximize the number of cells harvested.</li>
		<li>Use the plunger of a syringe to mash cells through the nylon membrane into the 50 ml conical tube to obtain a single cell suspension. Repeat with other mouse leg.</li>
	</ol>
	</li>
	<li>Centrifuge cells at 4 &#176;C for 10 min at 400 x g and discard supernatant. Resuspend BM in 5 ml ammonium chloride potassium (ACK) buffer to lyse red blood cells. Incubate cells on ice for 10 min.</li>
	<li>Fill tube with PBS to stop cell lysis. Centrifuge cells at 4 &#176;C for 10 min at 400 x g and discard supernatant. Repeat lysis step if the BM pellet has red color.</li>
	<li>Resuspend in 5 ml PBS and count cells by trypan blue exclusion using a hemocytometer.</li>
	<li>Resuspend cells at 2 x 106 cells per m with 2x pre-stim cocktail which contains DMEM supplemented with 20% FBS, L-glutamine, penicillin/streptomycin, IL-3 (14 ng/ml), IL-6 (24 ng/ml), and SCF (112 ng/ml). Plate 2 ml per well in 6 well plates.</li>
	<li>Incubate the cells overnight at 37 &#176;C and 5% CO2.</li>
	<li>Add 2 ml of viral supernatant to each well containing BM cells and add polybrene (8 &#181;g/ml). Spin plates at 30 &#176;C for 90 min at 1,500 x g then return plate to the tissue culture incubator overnight.</li>
	<li>Pipette cell suspension into conical tubes and centrifuge at 4 &#176;C for 10 min at 400 x g, do not discard the plate. Resuspend cells in 2 ml fresh 2x pre-stim cocktail per well and return to the original wells.</li>
	<li>Perform a 2nd spinoculation by repeating steps 3.7-3.9.</li>
</ol>
4. Transplant Donor Cells into Recipient Mice
<ol>
	<li>Prepare syngeneic recipient mice for transplant with lethal dose total body irradiation &#60; 24 hr prior to injection of BM cells. 
	NOTE: Lethal dose irradiation for BALB/c mice is typically 800-900 cGy and 1,000-1,200 cGy for C57B/610. The dose is typically split into two sessions of irradiation spaced at least 3-4 hours apart.</li>
	<li>Continued from step 3.11, harvest transduced donor cells from the 6 well plates by gently pipetting supernatant into conical tubes. Wash each well with 1 ml of PBS to collect residual cells and add to 50 ml conical tubes.</li>
	<li>Add 0.5 ml of 0.05% trypsin to wells and incubate at 37 &#176;C in the tissue culture incubator for 5 min.</li>
	<li>Add 1 ml D10 to wells and detach any remaining cells by gentle pipetting. Add cells to the respective tubes from step 4.2 and pellet by centrifugation at 4 &#176;C for 10 min at 400 x g.</li>
	<li>Wash cells with PBS and centrifuge at 4 &#176;C for 10 min at 400 x g. Resuspend in 5 ml PBS and count cells using a hemocytometer by trypan blue exclusion. 
	NOTE: If the vector has a GFP tag then quantify the percentage of GFP-positive cells with a flow cytometer.</li>
	<li>Resuspend 5 x 105 -&#160; 2 x 106 cells in 50 - 100 &#181;l PBS for injection into mice.</li>
	<li>Inject cells via retro-orbital injection into anesthetized mice using a sterile 27 G x &#189;&#34; needle. 
	NOTE: Other injection techniques include tail-vein, splenic, or femoral.
	<ol>
		<li>Anesthetize mouse using isoflurane and pinch foot to assess responsiveness.</li>
		<li>Position the mouse on its side and restrain by using both the thumb and index finger such that the eye protrudes slightly from the head.</li>
		<li>With the bevel facing up, insert the needle lateral to the medial canthus at a 45&#176; angle. 
		NOTE: If this technique is performed correctly, the eye should retract slightly and no resistance should be felt upon needle insertion.</li>
		<li>Examine mouse for any signs of injury such as swelling or bleeding. 
		NOTE: Following transplantation mice will have low blood counts for a number of weeks and so are susceptible to infection. Prophylaxis with antibiotics or use of acidified water may be used.</li>
	</ol>
	</li>
	<li>One month post-transplant, measure the % GFP in the peripheral blood as an indicator for engrafted donor cells by flow cytometry. 
	NOTE: GFP alone cannot determine success of engraftment of the cells. The viral transduction may have failed yet engraftment may have occurred. In this case, no GFP+ cells would be detected, but engraftment of the transplanted cells was successful.
	<ol>
		<li>Take 10 &#181;l of peripheral blood by puncturing the saphenous leg vein with a 21 G x 1 &#189;&#34; syringe and add it to 100 mM EDTA in PBS to prevent coagulation.</li>
		<li>Add 1 ml ACK to blood and lyse on ice for 10 min. Pellet cells by centrifugation at 400 x g for 10 min.</li>
		<li>Aspirate supernatant in a vacuum flask and add 1 ml PBS supplemented with 2% FBS to stop lysis. Pellet cells by centrifugation at 400 x g for 10 min.</li>
		<li>Aspirate supernatant in a vacuum flask and resuspend pellet in 100 &#181;l PBS supplemented with 2% FBS.</li>
		<li>Run samples on a flow cytometer and collect 300,000 events within the live cell gate (determined by FSC vs. SSC) and calculate the percentage of GFP+ and GFP- cells9.</li>
	</ol>
	</li>
	<li>Additionally at 1 month post-transplant, perform complete blood counts (CBC) using an automated veterinary hematology analyzer18 to monitor disease progression. 
	NOTE: Continue to monitor disease by % GFP in peripheral blood and CBC monthly.</li>
	<li>To terminate the experiment, anesthetize mouse using isoflurane and pinch foot to assess responsiveness. Sacrifice mouse by cervical dislocation and harvest spleen and bone marrow for histopathology. 
	NOTE: The length of the experiment is subjective and the termination date should be assessed based on the disease phenotype expected.</li>
</ol>

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The authors have nothing to disclose.

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><tbody><tr><td>&lt;strong&gt;Cell Lines&lt;/strong&gt;</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>&lt;strong&gt;Plasmids&lt;/strong&gt;</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 &amp;gamma;-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 &amp;gamma;-retroviral transfer plasmids are available from Addgene and others.</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>27 G x 1/2&quot; 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>10 cm 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>60 mm 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>100 &amp;#181;m 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>0.45 &amp;mu;m 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>&lt;strong&gt;Equipment&lt;/strong&gt;</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 ...

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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 ...

transduction-transplantation-mouse-model-myeloproliferative

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JoVE Journal

Cancer Research

11.8K Views.  University of California, Irvine. 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. 

Video: Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells (LSCs). An experimental AML rodent model to test therapies based on successfully transplanting these cells via retro-orbital injections in recipient mice is fraught with challenges. The aim of this study was to develop an easy, reliable, and consistent method to generate a robust murine model of AML using an intra-peritoneal route. In the present protocol, bone marrow cells were transduced with a retrovirus expressing human MLL-AF9 fusion oncoprotein. The efficiency of lineage negative (Lin-) and Lin-Sca-1+c-Kit+ (LSK) populations as donor LSCs in the development of primary AML was tested, and intra-peritoneal injection was adopted as a new method to generate AML. Comparison between intra-peritoneal and retro-orbital injections was done in serial transplantations to compare and contrast the two methods. Both Lin- and LSK&#160;cells transduced with human MLL-AF9 virus engrafted well in the bone marrow and spleen of recipients, leading to a full-blown AML. The intra-peritoneal injection of donor cells established AML in recipients upon serial transplantation, and the infiltration of AML cells was detected in the blood, bone marrow, spleen, and liver of recipients by flow cytometry, qPCR, and histological analyses. Thus, intra-peritoneal injection is an efficient method of AML induction using serial transplantation of donor leukemic cells.

Here, intra-peritoneal injection of leukemia cells is utilized to establish and propagate acute myeloid leukemia (AML) in mice. This new method is effective in the serial transplantation of AML cells and can serve as an alternative for those who may experience difficulties and inconsistencies with intravenous injection in mice.

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells ...

Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.

This protocol describes the isolation of double-negative thymocytes from the mouse thymus followed by retroviral transduction and co-culture on the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4) for further functional analysis.

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Immunology and Infection

Developing Efficient and Precise Mouse Model for Spinal Cord Glioma

Surgical Transplantation of Tumor Cells into the Spinal Cord of Mice

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and comprehensive data on spinal cord gliomas remain limited due to the lack of suitable preclinical animal models. Developing a simple and reproducible animal model has become essential for advancing basic and translational research. A murine model is ideal, as the murine spinal cord shares structural similarities with the human spinal cord. This protocol describes the generation of a reproducible murine model of spinal cord glioma by directly injecting tumor cells into the intervertebral space using the spinous process of the seventh cervical vertebra as a guide. Compared to other methods, this approach is more effective and convenient, involving a smaller incision, reduced invasiveness and blood loss, faster recovery, and more stable tumor formation. This model is expected to advance the understanding of disease mechanisms, optimize surgical strategies, and support the development of therapeutic drugs for spinal cord gliomas.

The present protocol describes the development of a reproducible murine model of spinal cord glioma by injecting tumor cells into the intervertebral space, offering a more effective and less invasive approach for research and therapeutic development.

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point ...

Neuroscience

A Technique to Generate a Human PBMC-Engrafted Humanized Xenograft Mouse Model

Source: Li, Z., et al. <a href="https://app.jove.com/v/59679">A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research.</a> J. Vis. Exp. (2019)
This video demonstrates an assay to generate a humanized xenograft mouse model. An immunocompromised mouse is injected with cyclophosphamide to cause immune cell depletion. Patient-derived tumor cells mixed with human peripheral blood mononuclear cells (PBMCs) in a hydrogel are subcutaneously injected into the mouse, forming a tumor with infiltrating T cells.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. All procedures involving animal ...

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

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