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 engraftment of cells expressing the gene of interest or even mouse death.
A critical factor to a successful transduction-transplantation model that is often overlooked is the quality of virus. Virus with high titers facilitates good transduction efficiency and thus a strong basis for disease, whereas using a virus associated with low titer will result in fewer cells expressing the gene of interest. Transduction efficiency can be enhanced by reagents that increase contact between virus and cells such as polybrene and retronectin. Transduction of HSCs is known to be difficult even with high concentrations of virus, ecotropic retrovirus only infects dividing cells. The purpose of &#34;pre-stim&#34; media (containing SCF, IL-3, and IL-6) is to induce cycling of quiescent HSCs. The lentiviral system could be used as an alternative approach if infection of non-dividing cells is necessary.
Another factor that can have an effect on virus generation is the type of transfection reagent used. Optimal transfection reagents should be determined empirically as marked differences in cell toxicity have been observed between research groups. The transfection reagent used here has resulted in good viral yield with low cell toxicity. Other common transfection reagents include calcium phosphate11 and nonliposomal transfection reagent such as Fugene. This protocol uses plasmid as the packaging vector with specific tissue trophisms for mouse and rat. If considering other animal models for transduction-transplantation, other packaging vectors with broader trophisms could also be used to generate amphotrophic (mouse, rat, human) or pantrophic (broad range trophisms) retrovirus.
An added advantage to the bone marrow transduction-transplantation model is the ability investigate the competitive advantage of two different donor populations or to express multiple genes of interest simultaneously by using unique markers to designate each population of interest. Such transplants often utilize congenic C57BL/6 mice to track donor cells beyond the use of an integrated GFP marker. Because congenic C57BL/6 mice can express leukocyte markers for either CD45.1 or CD45.2, donor cells can be obtained from one background and transplanted into the other. Moreover, a hybrid F1 strain can be generated to produce offspring expressing both CD45.1 and CD45.2 markers. Alternatively, the MSCV retroviral vector is available with several different tags, including unicistronic (e.g., FLAG HA) or bicistronic (e.g., GFP, RFP, YFP, mCherry, hCD4) constructs. These constructs may be used concurrently in competitive transplants to enable discrimination among donor populations and can be especially useful in situations where differentiation via CD45.1/CD45.2 expression is not available, such as the BALB/c mouse strain.
Another advantage of transduction-transplantation is the ability to investigate the contributions of specific cell types to disease initiation. While this protocol describes only the transduction-transplantation of whole bone marrow, other experimental designs may necessitate the enrichment or isolation of different HSC compartments. In these cases, the number of donor cells will vary according to the specific donor population used. Additionally, rescue cells from a na&#239;ve donor should be used as a supplement to support the mouse until HSC engraftment and expansion can occur. Table 2 outlines the recommended number of donor cells for transplantation of the various bone marrow compartments12-15.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td># of Cells / Transplant Recipient</td>
			<td># of Support Cells / Transplant Recipient</td>
		</tr>
		<tr>
			<td>Whole Bone Marrow</td>
			<td>500,000-2,000,000</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>c-Kit+</td>
			<td>1,000-10,000</td>
			<td>200,000</td>
		</tr>
		<tr>
			<td>Lin-c-Kit+Sca-1+ (LKS)</td>
			<td>100-10,000</td>
			<td>200,000</td>
		</tr>
		<tr>
			<td>LKS CD150+CD48- SP</td>
			<td>1-100</td>
			<td>200,000</td>
		</tr>
		<tr>
			<td>LKS CD150+CD34-</td>
			<td>1-100</td>
			<td>200,000</td>
		</tr>
	</tbody>
</table>
Table 2. Number of Donor Cells to Transplant&#8203;.
Furthermore, secondary transplant may also be performed to assess the ability of transduced cells to serially transplant disease.
Transduction-transplantation is a highly versatile method that is much faster and significantly more cost efficient as compared to transgenic, knock-in, or xenograft models. It allows one to quickly determine whether the gene of interest is sufficient to induce a hematologic malignancy, and can be used as an in vivo pre-clinical model to test drugs. Other benefits of the transduction-transplantation technique are that it avoids expression in non-hematopoietic cells and that the retroviral insertion site serves as a clonal marker. Limitations of this technique include non-physiological levels of transduced gene and differences in the integration site of the gene16,17. Taking all of the above mentioned benefits and limitations into account, transduction-transplantation is the obvious choice for initial in vivo modeling of putative hematopoietic oncogenes.

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 first month post-transplant. Evidence of a disease phenotype becomes apparent 3 months post-transplant where mice exhibit increased platelets and megakaryocytes, followed by bone marrow fibrosis at 6 - 10 months post-transplant. If donor cells fail to engraft, mouse death may occur within the first 2 weeks following transplant. In addition to poor injection or trauma during injection, other technical errors can cause poor engraftment of transduced cells or mouse death. These include sub-optimal irradiation of recipient mice, if this occurs most hematopoietic cells will be of recipient rather than donor origin. On the other hand, excessive irradiation of recipients can cause mice to develop irradiation-induced sickness or even death. Low viral titers will result in low infection efficiency of donor cells. As a consequence low numbers of donor cells will express the gene of interest and so mice may not demonstrate the desired phenotype. For this reason, it is crucial that virus with high titers is used to transduce donor cells. Figure 2 illustrates how to calculate relative viral titer where the % GFP is calculated from events collected from the live cell gate. Figure 2B illustrates a titer with more than one viral particle per cell (1:3 dilution) as well as titers with single viral particles per cell (1:30, 1:10, and 1:100 supernatant). Ideally, at least 50% GFP+ cells would be detected with 1:30 dilution of viral supernatant. Figure 3 shows evidence of a successful transplant recipient where CALRdel52 mice exhibit an MPN phenotype with increased platelets, increased megakaryocytes in the bone marrow, and bone marrow fibrosis.
<img alt="Figure 1" src="/files/ftp_upload/54624/54624fig1.jpg" /> 
Figure 1: Overview of Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasms. A) Retrovirus generation and determination of viral titer occurs 1 - 2 weeks prior to transplant. B) D(-8) (five days prior to bone marrow harvest), donor mice are treated with 5-FU to deplete lineage committed cells and induce hematopoietic stem cell cycling. C) On D(-3), bone marrow is harvested from leg bones and cultured in 2x pre-stim overnight to induce cell cycling of hematopoietic stem cells. D) Donor cells are infected with viral supernatant on D(-2) and the cells are centrifuged at 400 x g at 30 &#176;C for 1.5 hr for the 1st spinoculation. E) On D(-1), a 2nd spinoculation is performed on donor cells. Additionally, mice are irradiated to deplete cells in the host bone marrow niche to allow for donor cell engraftment. F) Transduced donor cells are transplanted into irradiated syngeneic recipient mice on D0. G) If donor cells fail to engraft then death will occur by day 12 - 14 following transplant if lethal dose irradiation has been used. H) The MPN phenotype becomes evident by 3 months post-transplant. <a href="https://www.jove.com/files/ftp_upload/54624/54624fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54624/54624fig2.jpg" /> 
Figure 2: Determination of Viral Titer by Flow Cytometry. 3T3 cells are transduced with different concentrations of virus and transduction efficiency is monitored via flow cytometry. A) Events are collected within the live cell gate as visualized by FSC vs. SSC. Dead cells are excluded from GFP analysis. B) Histograms show % GFP for each volume of viral supernatant tested. The viral titer is acceptable when at least 50% of 3T3 cells are GFP+ when infected with the 1:30 dilution of viral supernatant. Using the equation from step 2.11.6, the viral titer was calculated based on a cell count of 400,000 cells. The calculation for viral titer at a 1:30 dilution follows: [(400,000 x 0.56)/1) x 30] = 6.72 x 106 PFU/ml. Per step 3.7, infecting 4 x 106 cells with 2 ml of viral supernatant would result in each cell receiving 3 viral particles. <a href="https://www.jove.com/files/ftp_upload/54624/54624fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54624/54624fig3.jpg" /> 
Figure 3: Evidence of Successful Donor Cell Engraftment and MPN Phenotype. Irradiated recipient mice surviving beyond 2 weeks post-transplant indicates successful engraftment of injected cells. A) Transplanted donor cells can be monitored by % GFP in peripheral blood. B) Additionally, CBC counts should be taken to monitor disease progression. Increased platelets are detected as early as 2 months post-transplant in CALRdel52 when compared to CALRwt and empty vector control. C) At the time of termination, CALRdel52 mice exhibit classic features of the MPN phenotype including splenomegaly (not shown), bone marrow hypercellularity, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. <a href="https://www.jove.com/files/ftp_upload/54624/54624fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Technical Error</td>
			<td>Causes</td>
			<td>Result</td>
		</tr>
		<tr>
			<td>Poor Injection or Trauma</td>
			<td>Transplant Rejection</td>
			<td>Transplant Failure</td>
		</tr>
		<tr>
			<td>Bad Quality of Donor Cells</td>
			<td>Transplant Rejection</td>
			<td>Transplant Failure or Death</td>
		</tr>
		<tr>
			<td>Low Virus Titer</td>
			<td>Poor Transduction of HSC</td>
			<td>Transplant Failure</td>
		</tr>
		<tr>
			<td>High Irradiation Dose&#160;</td>
			<td>Irradiation induced sickness</td>
			<td>Death</td>
		</tr>
		<tr>
			<td>Low Irradiation Dose&#160;</td>
			<td>Transplant Rejection</td>
			<td>Transplant Failure</td>
		</tr>
		<tr>
			<td>Old Donor Mice (+12 Weeks)</td>
			<td>Low HSC Input</td>
			<td>Transplant Failure</td>
		</tr>
	</tbody>
</table>
Table 1: Technical Errors that can Result in Mouse Death or Transplant Failure. This table lists examples of technical errors and their associated causes that could occur with bone marrow transplant protocol.

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 ...

transduction-transplantation-mouse-model-myeloproliferative

Nanyang Technological University

Research

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

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor types, numerous mouse models have been developed in order to investigate the next generation of cancer therapies. However, in GIST, most in vivo studies use xenograft mouse models which have inherent limitations. Here, we describe an immunocompetent, genetically engineered mouse model of gastrointestinal stromal tumor harboring a KitV558&#916;/+ mutation. In this model, mutant KIT, the oncogene responsible for most GISTs, is driven by its endogenous promoter leading to a GIST which mimics the histological appearance and immune infiltrate seen in human GISTs. Furthermore, this model has been used successfully to investigate both targeted molecular and immune therapies. Here, we describe the breeding and maintenance of a KitV558&#916;/+ mouse colony. Additionally, this paper details the treatment and procurement of GIST, draining mesenteric lymph node, and adjacent cecum in KitV558&#916;/+ mice, as well as sample preparation for molecular and immunologic analyses.

The goal of this manuscript is to describe the KitV558&#916;/+&#160;mouse model and techniques for successful dissection and processing of mouse specimens.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor ...

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...