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In vivo Leukemogenesis Model Using Retrovirus Transduction
采用逆转录病毒转导的体内白血病生成模型   

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The Journal of Clinical Investigation
May 2017

Abstract

Various genetic alterations such as chromosomal translocation cause leukemia. For examples, gene rearrangements of the mixed-lineage leukemia (MLL) gene generate MLL fusion genes, whose products are potent oncogenic drivers in acute leukemia. To better understand the mechanism of disease onset, several murine leukemia models using retroviral gene transduction, xenograft, or Cre-mediated chromosomal translocation have been developed over the past twenty years. Particularly, a retroviral gene transduction-mediated murine leukemia model has been frequently used in the leukemia research field. Here, we describe the detailed protocol for this model.

Keywords: Mixed lineage leukemia (混合细胞系白血病), Leukemogenesis (白血病生成), MLL fusion (MLL融合), Leukemia (白血病), Mouse model (小鼠模型)

Background

Gene rearrangements generate mixed-lineage leukemia (MLL) fusion genes, which cause highly aggressive acute leukemia. MLL-rearrangements are often associated with few additional genetic alterations and poor clinical outcomes (Andersson et al., 2015). Wild-type MLL enhances and maintains the expression of a subset of genes, including homeobox (Hox) genes, to stimulate the expansion of immature progenitors (Jude et al., 2007). The expression of Hoxa9 and Meis1 is highest in the immature progenitor/stem cell fraction, but gradually declines as cells differentiate, and eventually diminishes in terminally-differentiated cell fractions (Somervaille and Cleary, 2006; Yokoyama et al., 2013). The MLL fusion protein constitutively up-regulates the expression of target genes, including Hoxa9 and Meis1, to immortalize immature progenitor cells and cause leukemia in vivo (Ayton and Cleary, 2003; Lavau et al., 1997). To date, more than 130 different MLL-rearrangements have been identified (Meyer et al., 2017). Two-thirds of MLL-rearranged leukemia cases are caused by fusion with a gene that is part of the AF4 family-ENL family-P-TEFb (AEP) complex (Yokoyama et al., 2010). The MLL fusion proteins constitutively form an MLL/AEP hybrid complex on the target chromatin (Okuda et al., 2014; Yokoyama et al., 2010), which further associates with the SL1 complex to activate RNA polymerase II-dependent transcription (Okuda et al., 2015 and 2016). AEP-mediated transactivation of MLL target genes transformed myeloid progenitors ex vivo, but did not cause leukemia in vivo, which suggested that other function is additionally required for in vivo leukemogenesis (Okuda et al., 2017). Recently, we showed that the ability to recruit the DOT1L complex is necessary to cause leukemia in vivo in addition to the ability to recruit AEP using in vivo leukemogenesis model. Thus, the combinatorial use of the in vivo leukemogenesis model and myeloid progenitor transformation assay is necessary to dissect the functional properties of oncogenes. In this protocol, we describe the in vivo leukemogenesis model using retroviral transduction in detail.

Materials and Reagents

  1. Pipette tips
  2. Tissue culture 10-cm dish (Greiner Bio One International, catalog number: 664160 )
  3. Collagen-coated tissue culture 10-cm dishes (Corning, catalog number: 354450 )
  4. 15-ml conical centrifuge tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339650 )
  5. Terumo syringe® 10-ml (Terumo, catalog number: SS-10ESZ )
  6. Terumo needle 21 G x1 ½” (Terumo, catalog number: NN-2138S )
  7. MS column (Miltenyi Biotec, catalog number: 130-042-201 )
  8. Pre-separation filter (Miltenyi Biotec, catalog number: 130-041-407 )
  9. Tissue culture 48-well plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150687 )
  10. Millex-GV 0.45-µm PVDF 33 mm sterile syringe filter (Merck, catalog number: SLHV033RB )
  11. Terumo® tuberculin syringe 26 G x ½” (Terumo, catalog number: SS-01T2613S )
  12. Terumo Myjector® insulin syringe 29 G x ½” (Terumo, catalog number: SS-05M2913 )
  13. Terumo syringe® 1-ml (Terumo, catalog number: SS-01T )
  14. Terumo needle 18 G x1 ½” (Terumo, catalog number: NN-1838S )
  15. Tissue culture 12-well plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150628 )
  16. Bottle-top filter 0.2-µm PVDF (Corning, catalog number: 431098 )
  17. Five-week-old female C57BL/6JJcl mice (donor mice) (CLEA Japan, Tokyo, Japan)
  18. Seven-week-old female C57BL/6JJcl mice (recipient mice) (CLEA Japan)
  19. Platinum-E packaging (PLAT-E) cell line (Morita et al., 2000) (gifted from Dr. Toshio Kitamura, or Cell Biolabs, catalog number: RV-101 )
  20. WEHI-3 (ATCC, catalog number: TIB-68 )
  21. pMSCV-neo vector (Takara Bio, ClontechTM, catalog number: 634401 )
  22. pMSCV-neo-MLL-ENL (request to Akihiko Yokoyama: ayokoyam@ncc-tmc.jp)
  23. Trypsin-EDTA solution (NACALAI TESQUE, catalog number: 32778-34 )
  24. LipofectamineTM 2000 transfection reagent (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  25. Opti-MEMTM I medium (Thermo Fisher Scientific, InvitrogenTM, catalog number: 31985070 )
  26. 70 % ethanol
  27. CD117 microbeads, mouse (Miltenyi Biotec, catalog number: 130-091-224 )
  28. 2.5% Baytril solution (Bayer, BaytrilTM, https://www.baytril.com/en/farm-animals/product/oral/)
  29. Polybrene infection/transfection Reagent (10 mg/ml) (Merck, catalog number: TR-1003-G )
  30. Bambanker serum-free freezing media (NIPPON Genetics, catalog number: BB01 )
  31. G418 solution (NACALAI TESQUE, catalog number: 16513-84 )
  32. Sodium chloride (NaCl) (NACALAI TESQUE, catalog number: 31320-05 )
  33. Na2HPO4·12H2O (Wako Pure Chemical Industries, catalog number: 196-02835 )
  34. Potassium dihydrogenphosphate (KH2PO4) (Wako Pure Chemical Industries, catalog number: 164-22635 )
  35. Potassium chloride (KCl) (Wako Pure Chemical Industries, catalog number: 160-22115 )
  36. Fetal bovine serum (FBS) (NICHIREI, Sigma-Aldrich, catalog number: 172012-500ml )
  37. Penicillin-streptomycin-glutamine (P/S) solution (NACALAI TESQUE, catalog number: 06168-34 )
  38. Dulbecco’s modified Eagle medium (DMEM) (NACALAI TESQUE, catalog number: 08459-64 )
  39. RPMI 1640 (NACALAI TESQUE, catalog number: 30264-56 )
  40. Ethylenediaminetetraacetic acid (EDTA) (NACALAI TESQUE, catalog number: 15130-95 )
  41. Sodium hydroxide (NaOH) (Wako Pure Chemical Industries, catalog number: 198-13765 )
  42. Ammonium chloride (NH4Cl) (Wako Pure Chemical Industries, catalog number: 017-02995 )
  43. Potassium hydrogen carbonate (KHCO3) (Wako Pure Chemical Industries, catalog number: 166-03275 )
  44. Bovine serum albumin (BSA) (Wako Pure Chemical Industries, catalog number: 019-23293 )
  45. Murine stem cell factor (SCF) (PeproTech, catalog number: 250-03 )
  46. Murine interleukin-3 (IL-3) (PeproTech, catalog number: 213-13 )
  47. Murine interleukin-6 (IL-6) (PeproTech, catalog number: 216-16 )
  48. Murine granulocyte-macrophage colony-stimulating factor (GM-CSF) (PeproTech, catalog number: 315-03 )
  49. IMDM powder (Thermo Fisher Scientific, GibcoTM, catalog number: 12200036 )
  50. Sodium hydrogen carbonate (NaHCO3) (Wako Pure Chemical Industries, catalog number: 195-14515 )
  51. Methyl cellulose (viscosity: 4,000 cP) (Sigma-Aldrich, catalog number: M0512 )
  52. Beta-mercaptoethanol (NACALAI TESQUE, catalog number: 21418-84 )
  53. Sodium pentobarbital (NACALAI TESQUE, catalog number: 02095-04 )
  54. 25x phosphate-buffered saline Ca2+/Mg2+-free (PBS) (see Recipes)
  55. D10 media (see Recipes)
  56. R10 media (see Recipes)
  57. R10W10 media (see Recipes)
  58. 0.5 M EDTA solution (see Recipes)
  59. ACK lysis buffer (see Recipes)
  60. SM buffer (see Recipes)
  61. Cytokine stocks (see Recipes)
  62. AC media (see Recipes)
  63. Anesthetic solution (see Recipes)

Equipment

  1. Pipetman P2, P20, P200, P1000
  2. 5% CO2 incubator 37 °C
  3. 5% CO2 incubator 32 °C
  4. Centrifuge for 15-ml conical tubes
  5. Cell counter (Hemacytometer)
  6. Laminar flow cabinet
  7. Surgical scissors and forceps
  8. MACS multistand (Miltenyi Biotec, catalog number: 130-042-303 )
  9. MiniMACS separator (Miltenyi Biotec, catalog number: 130-042-102 )
  10. Portable Pipet-Aid® XP pipette controller (Drummond Scientific, catalog number: 4-000-101 )
  11. -80 °C freezer
  12. 1-L glass bottle
  13. Autoclave
  14. Shaker
  15. Gammacell® 40 exactor with cesium-137 (Best Theratronics, model: Gammacell® 40 )
  16. Inverted microscope for general cell culture

Software

  1. GraphPad Prism (GraphPad Software, La Jolla, CA, USA)

Procedure

Schedule:
Day 1. Start culturing PLAT-E cells from freeze stock
Day 4. Replate PLAT-E cells for transfection
Day 5. Transfect PLAT-E cells
Day 6. Preparation of c-kit positive cells and irradiation of mice
Day 7. Transduction of retrovirus to c-kit positive cells and injection
Notes:

  1. Prepare a donor mouse (five-week-old female C57BL/6J mouse) and five recipient mice (seven-week-old female C57BL/6J mice) per sample.
  2. One positive control (MLL-ENL) and one negative control (Empty vector; pMSCV-neo) are required in this experiment.


  1. Virus preparation
    1. On day 1, thaw 4 x 106 PLAT-E cells from freeze stock and culture in 10-cm dishes with D10 media (see Recipes). The freezing media need to be removed for the initial culture.
      Note: Culture size depends on the sample size.
    2. On day 4, replate 4 x 106 PLAT-E cells in two 10-cm collagen-coated dishes with 10 ml of D10 media for a sample. In detail, remove the culture media, add 10 ml of PBS, remove PBS, add 1 ml of trypsin-EDTA and incubate for 5 min in a 37 °C 5% CO2 incubator. Add 9 ml of D10 media, transfer into a 15 ml tube, spin down cells at 400 x g for 5 min at room temperature, and then remove the supernatant. Add 10 ml of D10 media, count the cell number, and replate 4 x 106 PLAT-E cells in two 10-cm collagen-coated dishes with 10 ml of D10 media for a sample.
    3. On day 5, transfect PLAT-E cells with 16 µg of DNA and 40 µl of LipofectamineTM 2000 transfection reagent. At the time of transfection, the cell confluency should be 60-70%. In detail, dilute 16 µg of DNA in 1 ml of Opti-MEMTM media and dilute 40 µl of LipofectamineTM 2000 transfection reagent in 1 ml of Opti-MEMTM media, separately. After 5 min, combine the DNA solution with the LipofectamineTM 2000 solution. Mix gently and incubate for 20 min at room temperature. Add the DNA-Lipofectamine mixture to each dish and incubate in a 37 °C 5% CO2 incubator. After 6 h, replace the media with fresh D10 media and incubate in a 32 °C 5% CO2 incubator for 2 days.

  2. Preparation of c-kit (CD117) positive progenitor cells
    1. On day 6, euthanize the five-week-old female C57BL/6J mice by cervical dislocation, spray 70 % ethanol to the whole body in open air, and dissociate the femurs and tibiae from the donor mice in a laminar flow cabinet (Figure1). Peripheral muscle should be removed by surgical scissors as thoroughly as possible.
      Note: The volume of solution described in the following procedures is for the cells from one donor mouse. If the temperature is not specifically noted, all the procedures should be conducted at room temperature.


      Figure 1. Illustrative image of mouse tibia and femur

    2. Cut the end of the femurs and tibiae and flush the bone marrow with 10 ml of PBS (see Recipes) using a 10-ml syringe attached with a 21 G needle (Figure 2).


      Figure 2. Isolation of bone marrow cells from tibia and femur

    3. Homogenize the bone marrow cells gently by passing them through the 21 G needle several times.
    4. Spin down cells at 400 x g for 5 min, and then remove as much of the supernatant as possible.
    5. Resuspend the cells in 1 ml of ACK lysis buffer (see Recipes) and incubate on ice for 1 min.
    6. Add 10 ml of R10W10 media (see Recipes) and spin down cells at 400 x g for 5 min, and then remove the supernatant.
    7. Resuspend the cells in 10 ml of SM buffer (see Recipes) and spin down at 400 x g for 5 min, and then remove the supernatant.
    8. Resuspend the cells in 0.5 ml of SM buffer, add 20 µl of CD117 microbeads, and incubate for 20 min on ice or in the refrigerator.
    9. Add 10 ml of SM buffer and spin down cells at 400 x g for 5 min, and then remove the supernatant to wash the cells.
    10. Wash the cells again as in step B9.
    11. Install an MS column on a MACS magnetic stand and equilibrate with 1 ml of SM buffer.
    12. Place a pre-separation filter on top of the column and load the cells onto MS column through the pre-separation filter.
    13. Wash the column with 1 ml of SM buffer twice.
    14. Remove the column from the magnetic stand and place it on a new 15-ml conical tube.
    15. Add 1 ml of SM buffer to the column to elute c-kit-positive cells by gravity flow (optionally using a plunger).
    16. Add 10 ml of R10W10 media and spin down cells at 400 x g for 5 min, and then remove the supernatant.
    17. Resuspend all of the cells in 1 ml of R10W10 and transfer the cells into one well in a 48-well plate.
    18. Add cytokines (10 ng/ml SCF, 10 ng/ml IL-3, 10 ng/ml IL-6 at the final concentration) and incubate the cells in a 37 °C 5% CO2 incubator overnight.

  3. Irradiation of mice
    1. On day 7, irradiate recipient mice with a sub-lethal dose (6 Gy) in a gamma-cell. Several mice can be irradiated at the same time using an individually separatable cage.
    2. Add 1 ml of 2.5% Baytril solution to 200 ml of drinking water and treat for 1 week. The Baytril-containing water is not necessarily changed during the 1-week treatment.

  4. Virus transduction
    1. On day 7, count the cells. A total of 1-2 x 106 c-kit-positive cells are expected from a donor mouse.
    2. Add an appropriate amount of R10W10 media to make a 6 x 105 cell/ml suspension and add 1/250 volume of 10 mg/ml polybrene solution.
    3. Aliquot 1 ml of cell suspensions into 15-ml conical tubes (two tubes for each sample).
    4. Suck 10 ml of the virus supernatant from the PLAT-E cell culture using a 10-ml syringe, attach a filter (0.45-µm) to the 10-ml syringe, and add the virus supernatant directly to the c-kit-positive cell suspension.
    5. Spin the cell suspension at 1,100 x g for 2.5 h at 32 °C (Spinoculation).
    6. After spinoculation, remove the virus supernatant and wash the cells twice each with 10 ml of PBS.
    7. Resuspend the cells to 2 x 105 cells/100 µl in PBS.
      Note: The number of cells injected affects the duration and incidence of leukemogenesis. Thus, we can manipulate the duration by decreasing/increasing the cell number to some extent.
    8. Place the cell suspensions on ice until injection.

  5. Transplantation
    1. Inject 300 µl of anesthetic solution (see Recipes) intraperitoneally using a tuberculin syringe. Mice will be anesthetized completely within 5 min and stay asleep up to 1 h.
    2. After 5 min, inject 100 µl of the cell suspension into the retro-orbital sinus using a Myjector® insulin syringe. Pop up the eye, place the needle 1-2 mm deep between the eye and skin toward the brain to reach the vein, and gently inject the cell suspension (Figure 3) (Yardeni et al., 2011).
      Notes: Alternatively, the tail vein injection is applicable instead of retro-orbital injection.
    3. Place the mice into a cage for recovery and monitoring.


      Figure 3. Retro-orbital injection in mice

  6. Tumor cell isolation and culture
    1. When a mouse exhibits a morbid look, sacrifice the mouse, and dissociate the tumor cells from the femurs and tibiae as the same method of bone marrow preparation (steps B1-B6). Optionally, dissociate the spleen for pathological analysis.
      Notes: Morbid look indicates the condition of losing weight, bad coat of fur, and minimum reaction against stimulation e.g., sound and poking with a fingertip. It can be normally observed from day 60 to day 140 in the primary transplantation, and from day 20 to day 50 in the secondary transplantation.
    2. Prepare the bone marrow cell suspension with ACK buffer treatment described in steps B2-B6.
    3. Add 1 ml of R10W10 media and count the cell number.
    4. Add an appropriate amount of R10W10 media to make a 5 x 105 cells/ml suspension.
    5. Aliquot 200 µl of the cell suspension, add 1 ml of AC media (see Recipes) using a 1-ml syringe attached with an 18 G needle, and mix by vortexing. Optionally, the residual cells can be stocked. In detail, spin down cells at 400 x g for 5 min, and then remove the supernatant. Resuspend the cells in freezing media (We typically use the Bambanker freezing solution, but R10W10 plus 10% DMSO can be alternatively used.) and directly store at -80 °C. For long-term storage, storage tube should be transferred to liquid nitrogen from -80 °C freezer.
    6. Add 20 µl of G418 solution to eliminate normal cells and incubate in a 37 °C 5% CO2 incubator for 5 days.
    7. Transfer the cells into a 12-well plate using a 1-ml syringe with an 18 G needle and incubate in a 37 °C 5% CO2 incubator for several days. During the culture, prepare the recipient mice for secondary transplantation. If the media turns orange during culture because of excess cells, add an additional 1 ml of AC media with G418.
      Note: It is important to maintain good culture conditions. Overgrowth drastically affects the incidence of leukemia in the second transplantation.

  7. Secondary transplantation
    1. Collect cells with 10 ml of PBS, transfer to 15-ml conical tubes, and spin at 400 x g for 5 min.
    2. Remove the supernatant, resuspend the cells in 10 ml of PBS, and remove the debris to pass through a Pre-separation filter.
    3. Count the cell number and resuspend the cells to 2 x 105 cells/100 µl in PBS.
      Note: The number of cells injected affects the duration and incidence of leukemogenesis. Thus, we can manipulate the duration by decreasing/increasing the cell number to some extent.
    4. Place the cell suspension on ice.
    5. Inject 300 µl of anesthetic solution intraperitoneally using a tuberculin syringe.
    6. Inject 100 µl of the cell suspension into the retro-orbital sinus using a Myjector® insulin syringe.
    7. Place the mice into a cage for recovery and monitoring.
      Note: The duration of survival in secondary transplantation shortens compared to that in initial transplantation (Figure 4). It could be because leukemia cells need to acquire genetic/epigenetic alterations during the first disease onset. Or, the cancer stem/initiating population may be more enriched at the time of second transplantation compared to the initial transplantation. It is unclear at this point why the duration has been shortened dramatically in the second transplantation.


      Figure 4. Leukemogenic potential of MLL-ENL in this model

Data analysis

  1. We typically perform this experiment a minimum of two times using five recipient mice per sample. Total ten recipient mice per sample are needed for primary transplantation in two independent experiments. To confirm the leukemogenic ability, five recipient mice are typically transplanted with primary leukemia cells.
  2. For survival analysis, we use GraphPad Prism software. To compare two data sets, we use a Log-rank method. To compare three or more data sets, we use the Gehan-Breslow-Wilcoxon test.

Recipes

  1. 25x phosphate-buffered saline Ca2+/Mg2+-free (PBS) (1 L)
    1. Mix 200 g NaCl, 72.4 g Na2HPO4·12H2O, 5 g KH2PO4, and 5 g KCl
    2. Bring to 1 L with distilled H2O
    3. Autoclave for 20 min at 121 °C
    4. Dilute to 1x with distilled H2O for working solution
  2. D10 media
    Add 55 ml of FBS and 5.5 ml of P/S solution to 500 ml of DMEM
  3. R10 media
    Add 55 ml of FBS and 5.5 ml of P/S solution to 500 ml of RPMI 1640
  4. R10W10 media
    1. Culture WEHI-3 in R10 media to confluent
    2. When media turns orange, collect and filter (0.22 µm) the media
    3. Aliquot and store at -80 °C
    4. Add 55 ml of FBS, 55 ml of WEHI-3 culture media, 5.5 ml of P/S solution to 500 ml of RPMI 1640
  5. 0.5 M EDTA solution
    1. Weigh 93.06 g EDTA
    2. Bring to 1 L with distilled water and adjust pH to 8.0 with NaOH
  6. ACK lysis buffer
    1. Mix 8.29 g of NH4Cl and 1 g of KHCO3
    2. Bring to 1 L with distilled water
    3. Add 200 µl of 0.5 M EDTA solution
    4. Filter (0.2-µm) and store at 4 °C
  7. SM buffer
    1. Add 15 ml of FBS to 500 ml of 1x PBS
    2. Filter (0.2-µm) and store at 4 °C
  8. Cytokine stocks
    1. Dissolve 10 mg of BSA powder in 10 ml of PBS and filter (0.2-µm) (PBS + 0.1% BSA)
    2. Dissolve cytokines (SCF, IL-3, IL6, GM-CSF) to 50 µg/ml with PBS + 0.1% BSA
    3. Aliquot and store at -80 °C
  9. AC media
    1. Dissolve IMDM powder with 500 ml of distilled water, add 3 g NaHCO3, and filter (0.2-µm)
    2. Weigh 16 g of methyl cellulose in a 1-L glass bottle
    3. Autoclave methyl cellulose powder for 20 min at 121 °C
    4. Dissolve sterile methyl cellulose with 300 ml of sterile water and 500 ml of IMDM in a shaker overnight
    5. Add 200 ml of FBS and 7 µl of beta-mercaptoethanol
    6. Aliquot and store at -20 °C
    7. Before use, add 20 µl SCF, 20 µl IL-3, 20 µl GM-CSF, and 1 ml P/S solution
  10. Anesthetic solution
    1. Dilute sodium pentobarbital solution by ten-fold with PBS before use
    2. The final concentration of sodium pentobarbital should be 5 mg/ml

Acknowledgments

This study was supported by JSPS KAKENHI grants to H.O. (number 17H07379) and A.Y. (number 16H05337). This protocol is based on a previous report by Lavau et al. (1997). The authors declare no conflict of interest.

References

  1. Andersson, A. K., Ma, J., Wang, J., Chen, X., Gedman, A. L., Dang, J., Nakitandwe, J., Holmfeldt, L., Parker, M., Easton, J., Huether, R., Kriwacki, R., Rusch, M., Wu, G., Li, Y., Mulder, H., Raimondi, S., Pounds, S., Kang, G., Shi, L., Becksfort, J., Gupta, P., Payne-Turner, D., Vadodaria, B., Boggs, K., Yergeau, D., Manne, J., Song, G., Edmonson, M., Nagahawatte, P., Wei, L., Cheng, C., Pei, D., Sutton, R., Venn, N. C., Chetcuti, A., Rush, A., Catchpoole, D., Heldrup, J., Fioretos, T., Lu, C., Ding, L., Pui, C. H., Shurtleff, S., Mullighan, C. G., Mardis, E. R., Wilson, R. K., Gruber, T. A., Zhang, J., Downing, J. R. and St. Jude Children's Research Hospital-Washington University Pediatric Cancer Genome, P. (2015). The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias. Nat Genet 47(4): 330-337.
  2. Ayton, P. M., and Cleary, M. L. (2003). Transformation of myeloid progenitors by MLL oncoproteins is dependent on Hoxa7 and Hoxa9. Genes Dev 17: 2298-2307.
  3. Jude, C. D., Climer, L., Xu, D., Artinger, E., Fisher, J. K. and Ernst, P. (2007). Unique and independent roles for MLL in adult hematopoietic stem cells and progenitors. Cell Stem Cell 1(3): 324-337.
  4. Lavau, C., Szilvassy, S. J., Slany, R. and Cleary, M. L. (1997). Immortalization and leukemic transformation of a myelomonocytic precursor by retrovirally transduced HRX-ENL. EMBO J 16(14): 4226-4237.
  5. Meyer, C., Burmeister, T., Groger, D., Tsaur, G., Fechina, L., Renneville, A., Sutton, R., Venn, N. C., Emerenciano, M., Pombo-de-Oliveira, M. S., Barbieri Blunck, C., Almeida Lopes, B., Zuna, J., Trka, J., Ballerini, P., Lapillonne, H., De Braekeleer, M., Cazzaniga, G., Corral Abascal, L., van der Velden, V. H. J., Delabesse, E., Park, T. S., Oh, S. H., Silva, M. L. M., Lund-Aho, T., Juvonen, V., Moore, A. S., Heidenreich, O., Vormoor, J., Zerkalenkova, E., Olshanskaya, Y., Bueno, C., Menendez, P., Teigler-Schlegel, A., Zur Stadt, U., Lentes, J., Gohring, G., Kustanovich, A., Aleinikova, O., Schafer, B. W., Kubetzko, S., Madsen, H. O., Gruhn, B., Duarte, X., Gameiro, P., Lippert, E., Bidet, A., Cayuela, J. M., Clappier, E., Alonso, C. N., Zwaan, C. M., van den Heuvel-Eibrink, M. M., Izraeli, S., Trakhtenbrot, L., Archer, P., Hancock, J., Moricke, A., Alten, J., Schrappe, M., Stanulla, M., Strehl, S., Attarbaschi, A., Dworzak, M., Haas, O. A., Panzer-Grumayer, R., Sedek, L., Szczepanski, T., Caye, A., Suarez, L., Cave, H. and Marschalek, R. (2017). The MLL recombinome of acute leukemias in 2017. Leukemia.
  6. Morita, S., Kojima, T. and Kitamura, T. (2000). Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 7(12): 1063-1066.
  7. Okuda, H., Kanai, A., Ito, S., Matsui, H. and Yokoyama, A. (2015). AF4 uses the SL1 components of RNAP1 machinery to initiate MLL fusion- and AEP-dependent transcription. Nat Commun 6: 8869.
  8. Okuda, H., Kawaguchi, M., Kanai, A., Matsui, H., Kawamura, T., Inaba, T., Kitabayashi, I. and Yokoyama, A. (2014). MLL fusion proteins link transcriptional coactivators to previously active CpG-rich promoters. Nucleic Acids Res 42(7): 4241-4256.
  9. Okuda, H., Stanojevic, B., Kanai, A., Kawamura, T., Takahashi, S., Matsui, H., Takaori-Kondo, A. and Yokoyama, A. (2017). Cooperative gene activation by AF4 and DOT1L drives MLL-rearranged leukemia. J Clin Invest 127(5): 1918-1931.
  10. Okuda, H., Takahashi, S., Takaori-Kondo, A. and Yokoyama, A. (2016). TBP loading by AF4 through SL1 is the major rate-limiting step in MLL fusion-dependent transcription. Cell Cycle 15(20): 2712-2722.
  11. Somervaille, T. C. and Cleary, M. L. (2006). Identification and characterization of leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell 10(4): 257-268.
  12. Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M. and Hoogstraten-Miller, S. (2011). Retro-orbital injections in mice. Lab Anim (NY) 40(5): 155-160.
  13. Yokoyama, A., Ficara, F., Murphy, M. J., Meisel, C., Hatanaka, C., Kitabayashi, I. and Cleary, M. L. (2013). MLL becomes functional through intra-molecular interaction not by proteolytic processing. PLoS One 8(9): e73649.
  14. Yokoyama, A., Lin, M., Naresh, A., Kitabayashi, I. and Cleary, M. L. (2010). A higher-order complex containing AF4 and ENL family proteins with P-TEFb facilitates oncogenic and physiologic MLL-dependent transcription. Cancer Cell 17(2): 198-212.

简介

各种基因改变如染色体易位导致白血病。 例如,混合谱系白血病(MLL )基因的基因重排产生MLL 融合基因,其产物是急性白血病中有效的致癌驱动因子。 为了更好地理解疾病发生的机制,过去二十年来已经开发了几种使用逆转录病毒基因转导,异种移植或Cre介导的染色体易位的鼠白血病模型。 特别地,逆转录病毒基因转导介导的鼠白血病模型已经在白血病研究领域中经常使用。 在这里,我们描述这个模型的详细协议。

【背景】基因重排产生混合谱系白血病(MLL )融合基因,导致高度侵袭性的急性白血病。 MLL重排往往伴随着少量额外的遗传改变和不良的临床结果(Andersson等人,2015)。野生型MLL增强和维持包括同源框(Hox)基因在内的一部分基因的表达,以刺激未成熟祖细胞的扩增(Jude等人,2007年)。在未成熟的祖细胞/干细胞组分中,Hoxa9和emis1的表达最高,但随着细胞分化逐渐下降,并最终在终末分化的细胞组分中减少(Somervaille和Cleary,2006; Yokoyama et al。 ,2013)。 MLL融合蛋白组成性地上调包括Hoxa9和Meis1在内的靶基因的表达,使未成熟的祖细胞永生化,并在体内引起白血病 (Ayton和Cleary,2003; Lavau等人,1997)。迄今为止,已经确定了超过130种不同的MLL重新排列(Meyer等人,2017)。三分之二的MLL重新排列的白血病病例是由与AF4家族-ENL家族-P-TEFb(AEP)复合物(Yokoyama等人,,2010)。 MLL融合蛋白组成性地在靶染色质上形成MLL / AEP杂合复合物(Okuda等人,2014; Yokoyama等人,2010),其进一步与SL1复合物激活RNA聚合酶II依赖性转录(Okuda等人,2015和2016)。 AEP介导的MLL靶基因的反式激活转化了骨髓祖细胞体外,但是并没有引起体内白血病,这暗示体内其他功能额外需要 白血病发生(Okuda et al。,2017)。最近,我们显示除了使用体内白血病发生模型招募AEP的能力之外,招募DOT1L复合物的能力对于体内引起白血病是必要的。因此,体内白血病发生模型和骨髓祖细胞转化试验的组合使用对于解析致癌基因的功能特性是必需的。在该协议中,我们详细描述了使用逆转录病毒转导的体内白血病发生模型。

关键字:混合细胞系白血病, 白血病生成, MLL融合, 白血病, 小鼠模型

材料和试剂

  1. 移液器吸头
  2. 组织培养10厘米培养皿(Greiner Bio One International,目录号:664160)

  3. 胶原蛋白涂层组织培养10厘米的菜肴(康宁,目录号:354450)
  4. 15ml锥形离心管(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:339650)
  5. Terumo注射器10-ml(Terumo,目录号:SS-10ESZ)
  6. Terumo针21 G x1 1/2“(Terumo,目录号:NN-2138S)
  7. MS柱(Miltenyi Biotec,目录号:130-042-201)
  8. 预分离过滤器(Miltenyi Biotec,目录号:130-041-407)
  9. 组织培养48孔板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:150687)
  10. Millex-GV 0.45-μmPVDF 33毫米无菌注射器过滤器(Merck,目录号:SLHV033RB)
  11. Terumo结核菌素注射器26G×1/2“(Terumo,目录号:SS-01T2613S)
  12. Terumo Myjector胰岛素注射器29 G x 1/2“(Terumo,目录号:SS-05M2913)
  13. Terumo注射器1-ml(Terumo,目录号:SS-01T)
  14. Terumo针18 G x1½“(Terumo,目录号:NN-1838S)
  15. 组织培养12孔板(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:150628)
  16. 瓶顶过滤器0.2-μmPVDF(Corning,目录号:431098)
  17. 五周龄雌性C57BL / 6JJcl小鼠(供体小鼠)(CLEA日本,东京,日本)
  18. 七周龄雌性C57BL / 6JJcl小鼠(受体小鼠)(CLEA日本)
  19. Platinum-E包装(PLAT-E)细胞系(Morita等,2000)(由Toshio Kitamura博士或Cell Biolabs,产品目录号:RV-101提供)
  20. WEHI-3(ATCC,目录号:TIB-68)
  21. pMSCV-neo载体(Takara Bio,Clontech TM,目录号:634401)
  22. pMSCV-neo-MLL-ENL(请求横滨明彦: ayokoyam@ncc-tmc.jp
  23. 胰蛋白酶-EDTA溶液(NACALAI TESQUE,目录号:32778-34)
  24. Lipofectamine TM 2000转染试剂(Thermo Fisher Scientific,Invitrogen TM,目录号:11668019)
  25. Opti-MEM TM I培养基(Thermo Fisher Scientific,Invitrogen TM,目录号:31985070)。
  26. 70%乙醇
  27. CD117微珠,小鼠(Miltenyi Biotec,目录号:130-091-224)
  28. 2.5%Baytril溶液(Bayer,Baytril TM,, https ://www.baytril.com/en/farm-animals/product/oral/
  29. Polybrene感染/转染试剂(10 mg / ml)(Merck,目录号:TR-1003-G)
  30. Bambanker无血清冷冻培养基(NIPPON Genetics,目录号:BB01)
  31. G418解决方案(NACALAI TESQUE,目录号:16513-84)
  32. 氯化钠(NaCl)(NACALAI TESQUE,目录号:31320-05)
  33. Na 2 HPO 4•12H 2 O(Wako Pure Chemical Industries,目录号:196-02835)。
  34. 磷酸二氢钾(KH 2 PO 4)(Wako Pure Chemical Industries,目录号:164-22635)
  35. 氯化钾(KCl)(和光纯药工业,目录编号:160-22115)
  36. 胎牛血清(FBS)(NICHIREI,Sigma-Aldrich,目录号:172012-500ml)
  37. 青霉素 - 链霉素 - 谷氨酰胺(P / S)溶液(NACALAI TESQUE,目录号:06168-34)
  38. 达尔伯克改良伊格尔培养基(DMEM)(NACALAI TESQUE,目录号:08459-64)
  39. RPMI 1640(NACALAI TESQUE,目录号:30264-56)
  40. 乙二胺四乙酸(EDTA)(NACALAI TESQUE,目录号:15130-95)
  41. 氢氧化钠(NaOH)(和光纯药工业,目录号:198-13765)
  42. 氯化铵(NH4Cl)(Wako Pure Chemical Industries,目录号:017-02995)
  43. 碳酸氢钾(KHCO 3)(Wako Pure Chemical Industries,目录号:166-03275)
  44. 牛血清白蛋白(BSA)(Wako Pure Chemical Industries,目录号:019-23293)
  45. 鼠干细胞因子(SCF)(PeproTech,目录号:250-03)
  46. 鼠白细胞介素-3(IL-3)(PeproTech,目录号:213-13)
  47. 鼠白细胞介素-6(IL-6)(PeproTech,目录号:216-16)
  48. 鼠粒细胞巨噬细胞集落刺激因子(GM-CSF)(PeproTech,目录号:315-03)
  49. IMDM粉末(Thermo Fisher Scientific,Gibco TM,目录号:12200036)
  50. 碳酸氢钠(NaHCO 3)(Wako Pure Chemical Industries,目录号:195-14515)
  51. 甲基纤维素(粘度:4000cP)(Sigma-Aldrich,目录号:M0512)
  52. β-巯基乙醇(NACALAI TESQUE,目录号:21418-84)
  53. 戊巴比妥钠(NACALAI TESQUE,目录号:02095-04)
  54. 25X磷酸盐缓冲盐水Ca2 + / Mg2 + / PBS(见食谱)
  55. D10媒体(见食谱)
  56. R10媒体(见食谱)
  57. R10W10媒体(见食谱)
  58. 0.5M EDTA溶液(见食谱)
  59. ACK裂解缓冲液(见食谱)
  60. SM缓冲区(见食谱)
  61. 细胞因子库存(见食谱)
  62. 交流媒体(见食谱)
  63. 麻醉解决方案(见食谱)

设备

  1. 移液器P2,P20,P200,P1000
  2. 5%CO 2培养箱37℃
  3. 5%CO 2培养箱32°C
  4. 离心15毫升锥形管
  5. 细胞计数器(血细胞计数器)
  6. 层流柜
  7. 手术剪刀和钳子
  8. MACS多中心(Miltenyi Biotec,目录号:130-042-303)
  9. MiniMACS分离器(Miltenyi Biotec,目录号:130-042-102)
  10. Portable Pipet-Aid XP移液管控制器(Drummond Scientific,目录号:4-000-101)
  11. -80°C冰箱
  12. 1升玻璃瓶
  13. 高压灭菌器
  14. 摇床
  15. 具有铯-137的Gammacell40反应器(Best Theratronics,型号:Gammacell40)
  16. 一般细胞培养倒置显微镜

软件

  1. GraphPad Prism(GraphPad Software,La Jolla,CA,USA)

程序

附表:
第1天开始从冷冻原料培养PLAT-E细胞 第4天。Replate PLAT-E细胞转染
第5天。转染PLAT-E细胞
第6天c-kit阳性细胞的制备和小鼠的照射
第7天。逆转录病毒转导到c-kit阳性细胞和注射液 注意:

  1. 制备每个样品的供体小鼠(5周龄雌性C57BL / 6J小鼠)和5只受体小鼠(7周龄雌性C57BL / 6J小鼠)。
  2. 本实验需要一个阳性对照(MLL-ENL)和一个阴性对照(空载体; pMSCV-neo)。


  1. 病毒制备
    1. 在第1天,用冷冻原液融化4×10 6个PLAT-E细胞并用D10培养基在10cm培养皿中培养(参见食谱)。初始培养需要移除冷冻培养基。
      注意:文化大小取决于样本大小。
    2. 在第4天,在10毫升D10培养基中的两个10厘米胶原包被的培养皿中重新铺板4×10 6 PLAT-E细胞作为样品。详细地说,删除培养基,加入10毫升的PBS,删除PBS,加入1毫升胰蛋白酶-EDTA和孵化5分钟在37°C 5%CO 2培养箱。加入9ml的D10培养基,转移到15ml的试管中,在室温下400×g离心5分钟,然后除去上清液。加入10毫升的D10培养基,计数细胞数量,并用10毫升的D10培养基样品在两个10厘米胶原蛋白包被的培养皿中重新铺板4×10 6个PLAT-E细胞。 />
    3. 在第5天,用16μgDNA和40μlLipofectamine TM 2000转染试剂转染PLAT-E细胞。转染时,细胞汇合度应为60-70%。详细地,在1ml Opti-MEM TM培养基中稀释16μgDNA,并在1ml Opti-MEM中稀释40μlLipofectamine TM 2000转染试剂> TM媒体,分开。 5分钟后,将DNA溶液与Lipofectamine TM 2000溶液合并。轻轻混合并在室温下孵育20分钟。将DNA-Lipofectamine混合物添加到每个培养皿中,并在37℃,5%CO 2培养箱中孵育。 6小时后,用新鲜的D10培养基更换培养基,并在32°C 5%CO 2培养箱中培养2天。

  2. 制备c-kit(CD117)阳性祖细胞
    1. 在第6天,通过颈椎脱臼对5周龄雌性C57BL / 6J小鼠实施安乐死,在室内全身喷洒70%乙醇,并在层流柜中将供体小鼠的股骨和胫骨解离(图1) 。
      应尽可能彻底地用外科手术剪去除外周肌肉。
      注意:以下过程中描述的解决方案的体积是来自一个供体小鼠的细胞。如果温度没有特别说明,所有的程序应该在室温下进行。


      图1.小鼠胫骨和股骨的说明性图像

    2. 切割股骨和胫骨的末端,并用10毫升的PBS冲洗骨髓(见食谱),使用附带21 G针的10毫升注射器(图2)。


      图2.从胫骨和股骨中分离骨髓细胞


    3. 轻轻匀化骨髓细胞,使其通过21G针头
    4. 将细胞以400×g离心5分钟,然后尽可能多地除去上清液。
    5. 重悬细胞在1毫升的ACK裂解缓冲液(见食谱),并在冰上孵育1分钟。
    6. 加入10毫升R10W10培养基(见食谱),并将细胞在400×g下旋转5分钟,然后除去上清液。
    7. 将细胞重悬于10ml SM缓冲液中(参见食谱)并在400×g下旋转5分钟,然后除去上清液。
    8. 用0.5 ml SM缓冲液重悬细胞,加入20μlCD117微珠,在冰上或冰箱中孵育20分钟。
    9. 加入10ml的SM缓冲液并在400×g下离心5分钟,然后取出上清液洗涤细胞。
    10. 按照步骤B9再次清洗细胞。
    11. 在MACS磁架上安装MS列
    12. 从磁力架上取下柱子,放在新的15毫升锥形管上。
    13. 向柱中加入1 ml SM缓冲液,通过重力流动(任选使用活塞)洗脱c-kit阳性细胞。
    14. 加入10ml的R10W10培养基,并在400×g下离心5分钟,然后除去上清液。
    15. 重悬1毫升R10W10中的所有细胞,并将细胞转移到48孔板中的一个孔中。
    16. 加入细胞因子(10ng / ml SCF,10ng / ml IL-3,10ng / ml终浓度的IL-6)并将细胞在37℃5%CO 2培养箱过夜。

  3. 照射小鼠
    1. 在第7天,在γ-细胞中以亚致死剂量(6Gy)照射受体小鼠。
      几个小鼠可以同时使用单独分离的笼子进行照射
    2. 添加1毫升的2.5%的拜耳剂溶液到200毫升的饮用水中,并处理1周。在一周的治疗过程中,含有Baytril的水不一定会发生变化。

  4. 病毒转导
    1. 在第7天,计数细胞。预计来自供体小鼠的总共1-2×10 6个c-kit阳性细胞。
    2. 加入适量的R10W10培养基,制成6×10 5个细胞/ ml悬浮液,加入1/250体积的10mg / ml聚凝胺溶液。
    3. 将1毫升细胞悬液分装到15毫升锥形管中(每个样品两个管)。
    4. 用10ml注射器吸取10ml来自PLAT-E细胞培养物的病毒上清液,将过滤器(0.45μm)连接到10ml注射器上,并将病毒上清液直接加到c-kit阳性细胞暂停。

    5. 在32℃下将细胞悬浮液在1,100×g下旋转2.5小时(Spinoculation)。
    6. spinoculation后,删除病毒上清液,并用10毫升PBS洗两次细胞。
    7. 将细胞重悬于PBS中2×10 5个细胞/100μl。
      注:注射的细胞数量影响白血病发生的持续时间和发生率。因此,我们可以在一定程度上通过减少/增加细胞数量来控制持续时间。
    8. 将细胞悬液放在冰上,直到注射。

  5. 移植
    1. 用结核菌素注射器腹膜内注射300μl的麻醉溶液(见食谱)。
      小鼠将在5分钟内完全麻醉,并保持睡眠1小时。
    2. 5分钟后,使用Myjector胰岛素注射器将100μl细胞悬液注入眶后窦。将眼睛放开,将眼睛和皮肤之间1-2毫米深的针头朝着大脑放置到静脉,轻轻地注射细胞悬液(图3)(Yardeni et al。,2011 )。
      注意:或者,尾静脉注射适用,而不是眼眶后注射
    3. 将老鼠放入笼子中进行恢复和监测。


      图3.小鼠的眼球后注射

  6. 肿瘤细胞分离和培养
    1. 当小鼠表现出病态的外观时,以与骨髓制备相同的方法(步骤B1-B6),牺牲小鼠并从股骨和胫骨解离肿瘤细胞。可选地,分离脾进行病理分析。
      注意:病态的外观表明减肥,皮毛不好的状况,以及对刺激的最小反应,例如用指尖刺激和戳动。在第一次移植中从第60天到第140天可以正常观察,在第二次移植中从第20天到第50天可以正常观察到。

    2. 用步骤B2-B6中描述的ACK缓冲液处理制备骨髓细胞悬液
    3. 添加1毫升的R10W10媒体,并计算细胞数量。
    4. 加入适量的R10W10培养基,制成5×10 5个细胞/ ml悬浮液。
    5. 分装200μL的细胞悬液,添加1毫升交流媒体(见食谱)使用1毫升注射器连接18 G针,并涡流混合。可选地,残留细胞可以被储存。详细地,将细胞在400×g下旋转5分钟,然后除去上清液。在冷冻介质中重悬细胞(我们通常使用Bambanker冷冻溶液,但也可以使用R10W10加10%DMSO),并直接储存在-80°C。对于长期储存,储存管应从-80°C冷冻机转移到液氮中。
    6. 加入20μl的G418溶液以消除正常细胞,并在37℃5%CO 2培养箱中培养5天。
    7. 使用带有18G针的1-ml注射器将细胞转移至12孔板中,并在37℃的5%CO 2培养箱中孵育几天。在培养过程中,准备受体小鼠进行二次移植。如果由于细胞过多而使培养基变成橙色,则添加1ml含G418的AC培养基。
      注:保持良好的文化条件是重要的。过度生长大大影响了第二次移植中白血病的发病率。

  7. 二次移植
    1. 用10ml PBS收集细胞,转移到15ml锥形管中,并在400×g下旋转5分钟。
    2. 去除上清液,重悬细胞在10毫升的PBS,并删除碎片通过预分离过滤器。
    3. 计数细胞数量并将细胞重悬于PBS中2×10 5个细胞/100μl。
      注:注射的细胞数量影响白血病发生的持续时间和发生率。因此,我们可以在一定程度上通过减少/增加细胞数量来控制持续时间。
    4. 将细胞悬液放在冰上。

    5. 注射结核菌素注射器腹腔注射300μl的麻醉剂
    6. 用Myjector胰岛素注射器将100μl细胞悬液注入眶后窦。
    7. 将老鼠放入笼中进行恢复和监测。
      注:与初次移植相比,二次移植的存活时间缩短(图4)。这可能是因为白血病细胞在第一次发病时需要获得遗传/表观遗传改变。或者,与初次移植相比,在二次移植时癌症干细胞/起始群体可能更富集。目前还不清楚为什么在第二次移植中,持续时间已经大大缩短。


      图4.该模型中MLL-ENL的致白血病潜能

数据分析

  1. 我们通常每个样品使用五只受体小鼠至少进行两次这个实验。在两个独立实验中,每个样品共需要10个受体小鼠进行初级移植。为了确认白血病的发生能力,五个受体小鼠通常移植原代白血病细胞。
  2. 为了生存分析,我们使用GraphPad Prism软件。为了比较两个数据集,我们使用Log-rank方法。为了比较三个或更多的数据集,我们使用Gehan-Breslow-Wilcoxon测试。

食谱

  1. (PBS)(1L)的25x磷酸盐缓冲盐水Ca 2 + / Mg 2+ /
    1. 混合200g NaCl,72.4g Na 2 HPO 4•12H 2 O,5g KH 2 PO 4, 4克和5克KCl
    2. 用蒸馏水调至1L

    3. 在121°C高压灭菌20分钟
    4. 用蒸馏水稀释至1x以获得工作溶液
  2. D10媒体

    加入55毫升FBS和5.5毫升P / S溶液到500毫升DMEM中
  3. R10媒体
    加入55毫升的FBS和5.5毫升的P / S溶液到500毫升的RPMI 1640
  4. R10W10媒体
    1. 在R10培养基中培养WEHI-3至融合
    2. 当介质变成橙色时,收集并过滤介质(0.22μm)
    3. 分装和存储在-80°C
    4. 加入55毫升FBS,55毫升WEHI-3培养基,5.5毫升P / S溶液到500毫升RPMI 1640中。
  5. 0.5M EDTA溶液
    1. 称重93.06 g EDTA
    2. 用蒸馏水调至1升,用NaOH调节pH至8.0
  6. ACK裂解缓冲液
    1. 混合8.29克NH 4 Cl和1克KHCO 3。
    2. 用蒸馏水加1升
    3. 加200μl的0.5M EDTA溶液
    4. 过滤(0.2-μm)并在4°C储存
  7. SM缓冲区

    1. 加入15 ml FBS到500 ml 1x PBS中
    2. 过滤(0.2-μm)并在4°C储存
  8. 细胞因子库存
    1. 将10mg的BSA粉末溶解在10ml的PBS中并过滤(0.2-μm)(PBS + 0.1%BSA)
    2. 用PBS + 0.1%BSA将细胞因子(SCF,IL-3,IL6,GM-CSF)溶解到50μg/ ml。
    3. 分装和存储在-80°C
  9. AC媒体
    1. 用500毫升蒸馏水溶解IMDM粉末,加入3克NaHCO 3和过滤器(0.2微米)
    2. 在1升玻璃瓶中称量16克甲基纤维素

    3. 在121℃高压灭菌甲基纤维素粉20分钟

    4. 用300毫升无菌水和500毫升IMDM在摇床中溶解无菌甲基纤维素
    5. 加200毫升的FBS和7微升的β-巯基乙醇
    6. 分装和存储在-20°C
    7. 使用前,加20μLSCF,20μLIL-3,20μLGM-CSF和1ml P / S溶液
  10. 麻醉解决方案
    1. 使用前用PBS稀释10倍的戊巴比妥钠溶液
    2. 戊巴比妥钠的最终浓度应为5 mg / ml

致谢

这项研究得到了JSPS KAKENHI对H.O.的资助。 (编号17H07379)和A.Y. (编号16H05337)。该协议基于Lavau et al。(1997)的先前报道。作者宣称没有利益冲突。

参考

  1. Andersson,AK,Ma,J.,Wang,J.,Chen,X.,Gedman,AL,Dang,J.,Nakitandwe,J.,Holmfeldt,L.,Parker,M.,Easton,J.,Huether, R.,Kriwacki,R.,Rusch,M.,Wu,G.,Li,Y.,Mulder,H.,Raimondi,S.,Pounds,S.,Kang,G.,Shi,L.,Becksfort, J.,Gupta,P.,Payne-Turner,D.,Vadodaria,B.,Boggs,K.,Yergeau,D.,Manne,J.,Song,G.,Edmonson,M.,Nagahawatte,P., Wei,L.,Cheng,C.,Pei,D.,Sutton,R.,Venn,NC,Chetcuti,A.,Rush,A.,Catchpoole,D.,Heldrup,J.,Fioretos,T.,Lu Downing,JR和St. Jude儿童研究医院的研究人员发现了一种新的治疗方法,华盛顿大学儿科癌症基因组,P.(2015)。 婴儿MLL重排急性淋巴细胞白血病体细胞突变的情况 Nat Genet 47(4):330-337。
  2. Ayton,P.M。和Cleary,M.L。(2003)。 MLL癌蛋白对骨髓祖细胞的转化依赖于Hoxa7和Em > Hoxa9 。 Dev 17:2298-2307。
  3. Jude,C. D.,Climer,L.,Xu,D.,Artinger,E.,Fisher,J.K。和Ernst,P。(2007)。 MLL在成人造血干细胞和祖细胞中独特而独立的作用 细胞干细胞 1(3):324-337。
  4. Lavau,C.,Szilvassy,S.J。,Slany,R.和Cleary,M.L。(1997)。 逆转录病毒转导的HRX-ENL对骨髓单核细胞前体的永生化和白血病转化。 > EMBO J 16(14):4226-4237。
  5. Meyer,C.,Burmeister,T.,Groger,D.,Tsaur,G.,Fechina,L.,Renneville,A.,Sutton,R.,Venn,NC,Emerenciano,M.,Pombo-de-Oliveira, MS,Barbieri Blunck,C.,Almeida Lopes,B.,Zuna,J.,Trka,J.,Ballerini,P.,Lapillonne,H.,De Braekeleer,M.,Cazzaniga,G.,Corral Abascal,L. ,Van der Velden,VHJ,Delabesse,E.,Park,TS,Oh,SH,Silva,MLM,Lund-Aho,T.,Juvonen,V.,Moore,AS,Heidenreich,O.,Vormoor, Zerkalenkova,E.,Olshanskaya,Y.,Bueno,C.,Menendez,P.,Teigler-Schlegel,A.,Zur Stadt,U.,Lentes,J.,Gohring,G.,Kustanovich,A.,Aleinikova, O.,Schafer,BW,Kubetzko,S.,Madsen,HO,Gruhn,B.,Duarte,X.,Gameiro,P.,Lippert,E.,Bidet,A.,Cayuela,JM,Clappier, Alonso,CN,Zwaan,CM,van den Heuvel-Eibrink,MM,Izraeli,S.,Trakhtenbrot,L.,Archer,P.,Hancock,J.,Moricke,A.,Alten,J.,Schrappe,M. ,Stanulla,M.,Strehl,S.,Attarbaschi,A.,Dworzak,M.,Haas,OA,Panzer-Grumayer,R.,Sedek,L.,Szczepansk i,T.,Caye,A.,Suarez,L.,Cave,H.和Marschalek,R.(2017)。 2017年急性白血病的MLL重组。 白血病 。
  6. Morita,S.,Kojima,T。和Kitamura,T。(2000)。 Plat-E:一种高效稳定的逆转录病毒瞬时包装系统 Gene Ther 7(12):1063-1066。
  7. Okuda,H.,Kanai,A.,Ito,S.,Matsui,H.和Yokoyama,A。(2015)。 AF4使用RNAP1机制的SL1组分启动MLL融合和AEP依赖性转录。 a> Nat Commun 6:8869.
  8. Okuda,H.,Kawaguchi,M.,Kanai,A.,Matsui,H.,Kawamura,T.,Inaba,T.,Kitabayashi,I.和Yokoyama,A。(2014)。 MLL融合蛋白将转录共激活因子与之前活跃的富含CpG的启动子相连接。 Nucleic Acids Res 42(7):4241-4256。
  9. Okuda,H.,Stanojevic,B.,Kanai,A.,Kawamura,T.,Takahashi,S.,Matsui,H.,Takaori-Kondo,A。和Yokoyama,A。(2017)。 AF4和DOT1L合作基因激活驱动MLL重排的白血病 J Clin Invest 127(5):1918-1931。
  10. Okuda,H.,Takahashi,S.,Takaori-Kondo,A.和Yokoyama,A。(2016)。 AF4通过SL1加载TBP是MLL融合依赖性转录的主要限速步骤。 / a> Cell Cycle 15(20):2712-2722。
  11. Somervaille,T.C。和Cleary,M.L。(2006)。 小鼠MLL-AF9急性髓性白血病中白血病干细胞的鉴定和表征 Cancer Cell 10(4):257-268。
  12. Yardeni,T.,Eckhaus,M.,Morris,H. D.,Huizing,M.和Hoogstraten-Miller,S.(2011)。 老鼠的复古轨道注射 Lab Anim (纽约) 40(5):155-160。
  13. Yokoyama,A.,Ficara,F.,Murphy,M.J.,Meisel,C.,Hatanaka,C.,Kitabayashi,I.和Cleary,M.L。(2013)。 MLL通过分子内相互作用而不是通过蛋白水解加工而起作用 PLoS一个 8(9):e73649。
  14. Yokoyama,A.,Lin,M.,Naresh,A.,Kitabayashi,I.和Cleary,M.L。(2010)。 包含AF4和ENL家族蛋白与P-TEFb的高级复合物促进致癌和生理MLL-依赖的转录。 癌症细胞 17(2):198-212。
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引用:Okuda, H. and Yokoyama, A. (2017). In vivo Leukemogenesis Model Using Retrovirus Transduction. Bio-protocol 7(23): e2627. DOI: 10.21769/BioProtoc.2627.
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