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 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.
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.
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
2. Determine Relative Viral Titer by Flow Cytometry
3. Transduce Donor Bone Marrow Cells
4. Transplant Donor Cells into 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.
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 °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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Technical Error | Causes | Result |
Poor Injection or Trauma | Transplant Rejection | Transplant Failure |
Bad Quality of Donor Cells | Transplant Rejection | Transplant Failure or Death |
Low Virus Titer | Poor Transduction of HSC | Transplant Failure |
High Irradiation Dose | Irradiation induced sickness | Death |
Low Irradiation Dose | Transplant Rejection | Transplant Failure |
Old Donor Mice (+12 Weeks) | Low HSC Input | Transplant Failure |
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.
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 "pre-stim" 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 naïve 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.
# of Cells / Transplant Recipient | # of Support Cells / Transplant Recipient | |
Whole Bone Marrow | 500,000-2,000,000 | n/a |
c-Kit+ | 1,000-10,000 | 200,000 |
Lin-c-Kit+Sca-1+ (LKS) | 100-10,000 | 200,000 |
LKS CD150+CD48– SP | 1-100 | 200,000 |
LKS CD150+CD34– | 1-100 | 200,000 |
Table 2. Number of Donor Cells to Transplant.
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.
The authors have nothing to disclose.
This work is supported by the V Foundation Scholar (AGF) and the MPN Research Foundation (AGF).
CELL LINES | |||
DMEM | Corning | MT-10-013-CV | |
293T cells | ATCC | CRL-11268 | |
3T3 cells | ATCC | CRL-1658 | |
PLASMIDS | |||
EcoPak, also known as pCL-Eco | Addgene | 12371 | Retroviral packaging cell lines, such as EcoPack 2-293, may be used in place of the EcoPak plasmid and standard 293T cells. Additional γ-retrovirus envelope and packaging plasmids are available from Addgene and others. |
MSCV-IRES-GFP (MIG) | Addgene | 20672 | Additional γ-retroviral transfer plasmids are available from Addgene and others. |
CONSUMABLES | |||
27G x 1/2" needles | BD | 305620 | |
Fetal bovine serum | Corning | MT-35-010-CV | |
Penicillin/streptomycin/L-glutamine | Corning | MT-30-009-CI | |
Trypsin-EDTA (0.05%) | Corning | MT-25-052-CI | Can be homemade |
PBS | Corning | MT-21-031-CV | |
10cm dishes | Fisher | 172931 | |
15 ml conical tubes | Fisher | 12565268 | |
60mm dishes | Fisher | 150288 | |
Polybrene | Fisher | NC9840454 | |
5-FU | Fisher | A13456-06 | |
100um cell strainers | Fisher | 22363549 | |
50 ml conical tubes | Fisher | 12565270 | |
6-well plate | Fisher | 130184 | |
FACS tubes | Fisher | 14-959-5 | |
0.45um syringe filters | Fisher | 0974061B | |
Opti-MEM | Gibco | 31985-070 | |
ACK buffer | Lonza | 10-548E | Can be homemade |
Recombinant murine IL-3 | Peprotech | 213-13 | |
Recombinant murine IL-6 | Peprotech | 216-16 | |
Recombinant murine SCF | Peprotech | 250-03 | |
X-tremeGENE 9 | Roche | 6365809001 | Transfection reagent |
1.5 ml centrifuge tubes | USA Scientific | 1615-5500 | |
EQUIPMENT | |||
BD Accuri C6 | |||
X-ray irradiator |