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Myeloid Progenitor Transformation Assay
髓系祖细胞转化试验   

编审
Jia Li
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Abstract

Numerous oncogenes have been identified to cause leukemia. For example, chromosomal translocation generates various fusion genes of the mixed-lineage leukemia (MLL) gene and a partner gene in leukemia, whose gene products transform primary myeloid progenitors into an immortalized state. To characterize the transforming ability of leukemic oncogenes, researchers in the field have developed an ex vivo murine myeloid transformation assay using retroviral gene transduction and its protocol has been improved over the past 10 years. Here, we provide the detailed procedure for this assay.

Keywords: Mixed lineage leukemia(混合细胞系白血病), Transformation(转化), Myeloid(髓系), Progenitor(祖细胞), MLL fusion(MLL融合)

Background

Chromosomal translocation generates a variety of MLL fusion genes that cause leukemia (Meyer et al., 2017). The wild-type mixed lineage leukemia (MLL) protein functions as a transcriptional regulator that enhances the expression of a set of genes including homeobox (Hox) genes in hematopoietic immature progenitor cells (Jude et al., 2007). During normal hematopoiesis, Hox genes are expressed in the stem/progenitor cell fractions, but are transcriptionally down-regulated throughout differentiation (Somervaille and Cleary, 2006; Yokoyama et al., 2013); however, the MLL fusion protein constitutively up-regulates its target genes and blocks differentiation to establish an immortalized state under ex vivo culture conditions. Recently, we reported that the MLL-ENL and MLL-AF10 fusion proteins recruit AF4 to activate transcription and also recruit the DOT1L complex to maintain the transcription of the same target genes to efficiently transform hematopoietic progenitors (Okuda et al., 2017). To investigate the molecular mechanism of leukemogenesis, one can perform a myeloid progenitor transformation assay. By this assay, one can identify essential functional domains of an oncoprotein relatively easily at low cost compared to an in vivo leukemogenesis assay. In this assay, the leukemic oncogene is transduced by a retrovirus into primary murine hematopoietic progenitor cells derived from bone marrow and the transduced cells are cultured in semi-solid medium containing cytokines for the myeloid lineage (Lavau et al., 1997). Cells fully transformed by an oncogene can often be established as a cell line and cultured indefinitely ex vivo. Therefore, one can perform gene knockdown experiments of these immortalized cells by transducing an sh-RNA-carrying lentivirus. Alternatively, one can immortalize progenitors derived from a genetically engineered mouse carrying conditional knockout alleles with loxP sites, and knockout the gene of interest by activating Cre-recombinase. We have shown that the MLL fusion protein associates with various co-factors to form a functional complex. MENIN and lens epithelium-derived growth factor (LEDGF) are essential co-factors for leukemogenic activity (Yokoyama et al., 2005; Yokoyama and Cleary, 2008; Okuda et al., 2014). AF4 and ENL family proteins are the most frequent MLL-fusion partners, accounting for two-thirds of MLL-rearranged leukemia incidence (Huret et al., 2001), and form a biochemically stable complex with the SL1 complex and the P-TEFb elongation factor to activate transcription initiation and elongation (Yokoyama et al., 2010; Okuda et al., 2015; Okuda et al., 2016). MLL fusion proteins appear to activate transcription through the association with these cofactors in immortalized cells. In these previous studies, we used an sh-RNA-mediated knockdown strategy and Cre-mediated knockout strategy with MLL fusion-immortalized cells to demonstrate the importance of cofactors in leukemic transformation. Because this method allows us to generate cell lines immortalized with different oncogenes in a short period of time, the efficacy of drugs can be analyzed on various cell lines that are dependent on different oncogenes (Grembecka et al., 2012). Thus, this assay is a powerful tool for functional analysis of leukemic oncogenes and testing of drug efficacies.

Materials and Reagents

  1. Pipette tips
  2. Tissue culture 10-cm dish (Greiner Bio One International, catalog number: 664160 )
  3. Collagen-coated tissue culture 6-cm dishes (Corning, catalog number: 354401 )
  4. Terumo syringe® 10 ml (Terumo, catalog number: SS-10ESZ )
  5. Terumo needle 21 G x 1 ½” (Terumo, catalog number: NN-2138S )
  6. Pre-separation filter (Miltenyi Biotec, catalog number: 130-041-407 )
  7. MS column (Miltenyi Biotec, catalog number: 130-042-201 )
  8. 15-ml conical centrifuge tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339650 )
  9. Tissue culture 48-well plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150687 )
  10. Terumo syringe® 5 ml (Terumo, catalog number: SS-05SZ )
  11. Millex-GV 0.45-µm PVDF 33-mm sterile syringe filter (Merck, catalog number: SLHV033RB )
  12. Corning® Cell Lifter (Corning, catalog number: 3008 )
  13. Terumo syringe® 1 ml (Terumo, catalog number: SS-01T )
  14. Terumo needle 18 G x 1 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 (CLEA Japan, Tokyo, Japan)
  18. Platinum-E packaging (PLAT-E) cell line (Morita et al., 2000) (gifted from Dr. Toshio Kitamura, or Cell Biolabs, catalog number: RV-101 )
  19. WEHI-3 (ATCC, catalog number: TIB-68 )
  20. pMSCV-neo vector (Takara Bio, ClontechTM, catalog number: 634401 )
  21. LipofectamineTM 2000 transfection reagent (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  22. Opti-MEMTM I medium (Thermo Fisher Scientific, InvitrogenTM, catalog number: 31985070 )
  23. CD117 microbeads, mouse (Miltenyi Biotec, catalog number: 130-091-224 )
  24. G418 solution (NACALAI TESQUE, catalog number: 16513-84 )
  25. Polybrene infection/transfection reagent (10 mg/ml) (Merck, catalog number: TR-1003-G )
  26. Beta-mercaptoethanol (NACALAI TESQUE, catalog number: 21418-84 )
  27. RNeasy® mini kit (QIAGEN, catalog number: 74106 )
  28. Superscript® III first-strand synthesis system for RT-PCR (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18080051 )
  29. Sodium chloride (NaCl) (NACALAI TESQUE, catalog number: 31320-05 )
  30. Na2HPO4·12H2O (Wako Pure Chemical Industries, catalog number: 196-02835 )
  31. Potassium dihydrogenphosphate (KH2PO4) (Wako Pure Chemical Industries, catalog number: 164-22635 )
  32. Potassium chloride (KCl) (Wako Pure Chemical Industries, catalog number: 160-22115 )
  33. Fetal bovine serum (FBS) (NICHIREI, Sigma-Aldrich, catalog number: 172012-500ML )
  34. Penicillin-streptomycin-glutamine (P/S) solution (NACALAI TESQUE, catalog number: 06168-34 )
  35. Dulbecco’s modified Eagle medium (DMEM) (NACALAI TESQUE, catalog number: 08459-64 )
  36. RPMI 1640 (NACALAI TESQUE, catalog number: 30264-56 )
  37. Ethylenediaminetetraacetic acid (EDTA) (NACALAI TESQUE, catalog number: 15130-95 )
  38. Trypsin-EDTA solution (NACALAI TESQUE, catalog number: 32778-34 )
  39. Sodium hydroxide (NaOH) (Wako Pure Chemical Industries, catalog number: 198-13765 )
  40. Ammonium chloride (NH4Cl) (Wako Pure Chemical Industries, catalog number: 017-02995 )
  41. Potassium hydrogen carbonate (KHCO3) (Wako Pure Chemical Industries, catalog number: 166-03275 )
  42. Murine stem cell factor (SCF) (PeproTech, catalog number: 250-03 )
  43. Murine interleukin-3 (IL-3) (PeproTech, catalog number: 213-13 )
  44. Murine interleukin-6 (IL-6) (PeproTech, catalog number: 216-16 )
  45. Murine granulocyte-macrophage colony-stimulating factor (GM-CSF) (PeproTech, catalog number: 315-03 )
  46. Bovine serum albumin (BSA) (Wako Pure Chemical Industries, catalog number: 019-23293 )
  47. IMDM powder (Thermo Fisher Scientific, GibcoTM, catalog number: 12200036 )
  48. Sodium hydrogen carbonate (NaHCO3) (Wako Pure Chemical Industries, catalog number: 195-14515 )
  49. Methyl cellulose (viscosity: 4,000 cP) (Sigma-Aldrich, catalog number: M0512 )
  50. TaqMan® probes against murine Hoxa9 and Gapdh
    Hoxa9 (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: Mm00439364_m1 )
    Gapdh (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: Mm99999915_g1 )
  51. TaqMan® fast advanced master mix (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4444557 )
  52. 25x phosphate-buffered saline Ca2+/Mg2+-free (PBS) (see Recipes)
  53. D10 media (see Recipes)
  54. R10 media (see Recipes)
  55. R10W10 media (see Recipes)
  56. 0.5 M EDTA solution (see Recipes)
  57. ACK lysis buffer (see Recipes)
  58. SM buffer (see Recipes)
  59. Cytokine stocks (see Recipes)
  60. AC media (see Recipes)

Equipment

  1. 5% CO2 incubator 37 °C
  2. 5% CO2 incubator 32 °C
  3. MACS multistand (Miltenyi Biotec, catalog number: 130-042-303 )
  4. Pipetman P2, P20, P200, P1000
  5. Portable Pipet-Aid® XP pipette controller (Drummond Scientific, catalog number: 4-000-101 )
  6. Microscope
  7. Cell counter
  8. Autoclave
  9. 1-L glass bottle
  10. Shaker
  11. Surgical scissors and forceps
  12. Laminar flow cabinet
  13. Centrifuge for 15-ml conical tubes
  14. MiniMACS separator (Miltenyi Biotec, catalog number: 130-042-102 )
  15. StepOnePlusTM realtime PCR system (Thermo Fisher Scientific, Applied BiosystemsTM, model: StepOnePlusTM , catalog number: 4376600)
  16. General equipment for Western blotting and SDS-PAGE

Software

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

Procedure

Schedule (see Figure 1):
Day 1. Start culturing PLAT-E cells from frozen stock
Day 3. Replate PLAT-E cells for transfection
Day 4. Transfect PLAT-E cells
Day 5. Preparation of c-kit-positive cells
Day 6. Transduction of retrovirus into c-kit-positive cells
Day 7. Addition of G418
Day 12. 1st passage
Day 17. 2nd passage
Day 22. Colony counting and 3rd passage
Day 27. Colony counting


Figure 1. Schedule of murine myeloid progenitor transformation assay

  1. Virus preparation
    1. On day 1, thaw PLAT-E cells from frozen stock and culture 4 x 106 cells in 10-cm dishes with 10 ml of D10 media (see Recipes) in a 37 °C 5% CO2 incubator.
    2. On day 3, replate 8 x 105 PLAT-E cells in 6-cm collagen-coated dishes with 5 ml of D10 media in a 37 °C 5% CO2 incubator (one positive control [MLL-ENL], one negative control [empty vector; pMSCV-neo], two mock controls for G418 selection, and samples [up to 16 samples]).
    3. On day 4, transfect PLAT-E cells with 8 µg of DNA and 20 µl of LipofectamineTM 2000 transfection reagent. At the time of transfection, the cell confluency should be 60-70%. (In detail, dilute 8 µg of DNA in 500 µl of Opti-MEMTM media and dilute 20 µl of LipofectamineTM 2000 transfection reagent in 500 µl 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.
      Note: Virus supernatant should be freshly prepared in every experiment because retrovirus is unstable for storage.

  2. Preparation of c-kit (CD117)-positive progenitor cells
    1. On day 5, harvest the femurs and tibiae and remove the peripheral muscle as thoroughly as possible from a five-week-old C57BL/6J mouse (Figure 2A).
    2. Cut the end of femurs and tibiae and flush bone marrow with 10 ml of PBS (see Recipes) using a 10-ml syringe attached with a 21 G needle (Figure 2B) (Video 1).

      Video 1. Bone marrow isolation from tibiae by flushing with PBS

    3. Homogenize the bone marrow cells gently by passing through a 21 G needle several times.
    4. Spin down the cells at 400 x g for 5 min at room temperature (Figure 2C left) 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 for 1 min on ice.
    6. Add 10 ml of R10W10 media (see Recipes) and spin down cells at 400 x g for 5 min at room temperature, and then remove the supernatant.
    7. Resuspend the cells in 10 ml of SM buffer (see Recipes) and spin down cells at 400 x g for 5 min at room temperature (Figure 2C right), 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 at room temperature, 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 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 the MS column through the pre-separation filter (Figure 2D).


      Figure 2. Representative image of c-kit-positive progenitor cell isolation. A. Harvested tibia and femur; B. Flushing the bone marrow with PBS; C. Red blood cell removal by ACK buffer treatment; D. c-kit-positive cell isolation by MACS.

    13. Wash the column twice with 1 ml of SM buffer.
    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 the cells at 400 x g for 5 min at room temperature, and then remove the supernatant.
    17. Resuspend all of the cells in 1 ml of R10W10 and transfer 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. Virus transduction and cell culture
    1. On day 6, count the cells. A total of 1-2 x 106 c-kit-positive cells are expected from each mouse.
    2. Add an appropriate amount of R10W10 media to prepare a 1 x 105 cells/ml suspension and add a 1/250 volume of 10 mg/ml polybrene solution.
    3. Aliquot 0.5 ml of cell suspensions into 15-ml conical tubes (additionally, prepare two mock infection controls).
    4. Suck 5 ml of the virus supernatant using a 5-ml syringe, attach a filter (0.45-µm) to the 5-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. During spinoculation, add 1 ml of PBS onto PLAT-E cells, harvest the cells using a cell lifter, and transfer the cells into a new tube.
    7. Wash the packaging cells with 1 ml of PBS again and prepare the whole cell lysate in 500 µl of WB lysis buffer.
    8. After spinoculation, remove the virus supernatant (leaving approximately 200 µl of media), resuspend the cells in residual media, add 1 ml of AC media (see Recipes) using a 1-ml syringe attached with an 18 G needle, and mix by vortexing.
      Note: Use a 1-ml syringe and a needle to take 1 ml of AC media because this media is very viscous. A pipet cannot be used.
    9. Incubate the 15-ml conical tubes containing the cells in a 37 °C 5% CO2 incubator overnight.
    10. On day 7, add 20 µl of G418 solution, mix by vortexing, and transfer the cells into a 12-well plate using a 1-ml syringe attached with an 18 G needle. For the two mock controls, add G418 in one tube to ensure that G418 selection is working.
      Note: Samples should be placed in the central 6 wells in a 12-well plate and PBS should be added to the peripheral 6 wells to prevent the cultures in AC media from drying.
    11. Incubate in a 37 °C 5% CO2 incubator for 5 days (Figure 3). If 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 colony formation in the next round of culture.
    12. Optionally, one can check the protein expression of transgenes in the packaging cells by WB during the first round of culture.


      Figure 3. Image of colonies during the 1st and 4th rounds of culture

  4. 1st passage
    1. On day 12, collect cells with 10 ml of PBS, transfer to a 15-ml conical tube, and spin down at 400 x g for 5 min at room temperature.
    2. Wash the cells with 10 ml of PBS.
      Note: It is important to suspend thoroughly by vigorous pipetting to remove the debris of AC media.
    3. Resuspend the cells in 1 ml of R10W10 media and count the cells.
    4. Transfer 4 x 104 cells to a new 15-ml conical tube and add an appropriate amount of R10W10 to prepare a 200-µl cell suspension.
    5. Add 1 ml of AC media and mix by vortexing. Transfer the cells into a well in a new 12-well plate using a 1-ml syringe attached with an 18 G needle.
      Note: Samples should be placed in the central 6 wells in a 12-well plate and PBS should be added to the peripheral 6 wells to prevent the cultures in AC media from drying.
    6. Incubate the cells in a 37 °C 5% CO2 incubator for 5 days. If the media turns orange during culture, add an additional 1 ml of AC media to the well.
    7. Optionally, collect residual cells into a new tube and lyse the cells in RLT lysis buffer containing 1% beta-mercaptoethanol of an RNeasy® mini kit for RNA isolation as described in Procedure E.

  5. Cell collection and analysis
    1. Isolate the RNA using the RNeasy® mini kit and synthesize cDNA using the Superscript® III first-strand synthesis system with oligo-dT primers.
    2. Evaluate gene expression by quantitative PCR. In case of myeloid progenitor transformation by MLL-fusion genes, Hoxa9 gene expression is a good indicator of immortalization.

  6. 2nd passage
    1. On day 17, harvest and count the cells as in 1st passage.
    2. Culture 2 x 104 cells/well.

  7. Colony counting and 3rd passage
    1. On day 22, count the colony number under a microscope.
      Note: No colonies should be observed in the vector control. It is important to set a standard which defines a colony and use this definition throughout the study. We typically consider a cluster of more than 100 cells as a colony.
    2. Harvest and count the cells as in the 1st passage.
    3. Culture 1 x 104 cells/well.

  8. Colony counting
    On day 27, count the colony number under a microscope (Figure 3).

Data analysis

  1. Because of experimental variations, this assay must be performed a minimum of three times.
  2. For statistical analysis, we use GraphPad Prism software. To compare two data sets, we perform Student’s t-test. To compare three or more data sets, we perform one-way analysis of variance and post-hoc Tukey correction.

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 confluence
    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, and 5.5 ml of P/S solution to 500 ml of RPMI 1640
  5. 0.5 M EDTA solution
    1. Weigh 93.06 g of 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 cytokines (SCF, IL-3, IL6, GM-CSF) to 50 µg/ml in PBS + 0.1% BSA
    2. Aliquot and store at -80 °C
  9. AC media (alternatively Methocult M3231 from STEMCELL Technologies, Vancouver, Canada)
    1. Dissolve IMDM powder in 500 ml of distilled water, add 3 g of 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 in 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 100 ml per bottle 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 to 100 ml of media

Acknowledgments

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

References

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  2. Huret, J. L., Dessen, P. and Bernheim, A. (2001). An atlas of chromosomes in hematological malignancies. Example: 11q23 and MLL partners. Leukemia 15(6): 987-989.
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简介

许多致癌基因已被确定为导致白血病。 例如,染色体易位在白血病中产生混合谱系白血病(MLL )基因和伴侣基因的各种融合基因,其基因产物将原始骨髓祖细胞转化为永生状态。 为了表征白血病癌基因的转化能力,本领域的研究人员已经利用逆转录病毒基因转导开发了一种离体小鼠骨髓转化试验,其方案在过去的10年中得到了改进。 在这里,我们提供了这个检测的详细程序。

【背景】染色体易位产生多种导致白血病的MLL融合基因(Meyer等人,2017)。野生型混合谱系白血病(MLL)蛋白质起着转录调节剂的作用,其在造血不成熟祖细胞中增强包括同源框(homoxox(Hox))基因在内的一组基因的表达(Jude等。,2007)。在正常造血期间,Hox基因在干/祖细胞部分中表达,但在分化过程中转录下调(Somervaille和Cleary,2006; Yokoyama等人, 2013年);然而,MLL融合蛋白组成性地上调其靶基因并阻断分化以在离体培养条件下建立永生状态。最近,我们报道了MLL-ENL和MLL-AF10融合蛋白招募AF4以激活转录并且还募集DOT1L复合物以维持相同靶基因的转录以有效地转化造血祖细胞(Okuda等人, 2017)。为了研究白血病发生的分子机制,可以进行骨髓祖细胞转化测定。通过该测定,与体内白血病发生测定相比,可以相对容易地以低成本鉴定癌蛋白的基本功能结构域。在该测定中,将白血病癌基因通过逆转录病毒转导到源自骨髓的原代鼠造血祖细胞中,并将转导的细胞培养在含有用于骨髓谱系的细胞因子的半固体培养基中(Lavau et al。,1997)。通过癌基因完全转化的细胞通常可以建立为细胞系并且无限期地体外培养。因此,可以通过转导携带sh-RNA的慢病毒进行这些永生化细胞的基因敲低实验。或者,可以将源自携带有条件敲除等位基因的基因工程小鼠的loxP位点的祖细胞永生化,并通过激活Cre重组酶来敲除感兴趣的基因。我们已经证明,MLL融合蛋白与各种辅因子结合形成功能性复合物。 MENIN和晶状体上皮衍生生长因子(LEDGF)是白血病发生活性的基本辅助因子(Yokoyama等人,2005; Yokoyama和Cleary,2008; Okuda等人 >,2014)。 AF4和ENL家族蛋白是最常见的MLL融合伴侣,占MLL重新安置的白血病发病率的三分之二(Huret等人,2001)和形式与SL1复合物和P-TEFb延伸因子形成生物化学稳定的复合物以激活转录起始和延伸(Yokoyama等人,2010; Okuda等人,2015; Okuda等人,2016年)。 MLL融合蛋白似乎通过与永生化细胞中的这些辅因子的缔合来激活转录。在以前的这些研究中,我们使用sh-RNA介导的敲低策略和Cre介导的敲除策略与MLL融合永生化细胞来证明辅因子在白血病转化中的重要性。因为这种方法使得我们能够在短时间内产生不同癌基因永生化的细胞系,所以可以在依赖于不同癌基因的各种细胞系上分析药物的功效(Grembecka等人, 2012)。因此,该测定法是白血病癌基因功能分析和药物功效测试的有力工具。

关键字:混合细胞系白血病, 转化, 髓系, 祖细胞, MLL融合

材料和试剂

  1. 移液器提示
  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6厘米的菜肴(康宁,目录号:354401)
  4. Terumo注射器10ml(Terumo,目录号:SS-10ESZ)
  5. Terumo针21 G x1½“(Terumo,目录号:NN-2138S)
  6. 预分离过滤器(Miltenyi Biotec,目录号:130-041-407)
  7. MS柱(Miltenyi Biotec,目录号:130-042-201)
  8. 15ml锥形离心管(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:339650)
  9. 组织培养48孔板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:150687)
  10. Terumo注射器5ml(Terumo,目录号:SS-05SZ)
  11. Millex-GV 0.45-μmPVDF 33-mm无菌注射器过滤器(Merck,产品目录号:SLHV033RB)
  12. Corning Cell Lifter(Corning,目录号:3008)
  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(CLEA日本,东京,日本)
  18. 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)
  19. WEHI-3(ATCC,目录号:TIB-68)
  20. pMSCV-neo载体(Takara Bio,Clontech TM,目录号:634401)
  21. Lipofectamine TM 2000转染试剂(Thermo Fisher Scientific,Invitrogen TM,目录号:11668019)
  22. Opti-MEM TM I培养基(Thermo Fisher Scientific,Invitrogen TM,目录号:31985070)。
  23. CD117微珠,小鼠(Miltenyi Biotec,目录号:130-091-224)
  24. G418解决方案(NACALAI TESQUE,目录号:16513-84)
  25. Polybrene感染/转染试剂(10 mg / ml)(Merck,目录号:TR-1003-G)
  26. β-巯基乙醇(NACALAI TESQUE,目录号:21418-84)
  27. RNeasy®mini试剂盒(QIAGEN,目录号:74106)
  28. 用于RT-PCR的Superscript III第一链合成系统(Thermo Fisher Scientific,Invitrogen TM,产品目录号:18080051)
  29. 氯化钠(NaCl)(NACALAI TESQUE,目录号:31320-05)
  30. Na 2 HPO 4•12H 2 O(Wako Pure Chemical Industries,目录号:196-02835)。
  31. 磷酸二氢钾(KH 2 PO 4)(Wako Pure Chemical Industries,目录号:164-22635)
  32. 氯化钾(KCl)(和光纯药工业,目录编号:160-22115)
  33. 胎牛血清(FBS)(NICHIREI,Sigma-Aldrich,目录号:172012-500ML)
  34. 青霉素 - 链霉素 - 谷氨酰胺(P / S)溶液(NACALAI TESQUE,目录号:06168-34)
  35. 达尔伯克改良伊格尔培养基(DMEM)(NACALAI TESQUE,目录号:08459-64)
  36. RPMI 1640(NACALAI TESQUE,目录号:30264-56)
  37. 乙二胺四乙酸(EDTA)(NACALAI TESQUE,目录号:15130-95)
  38. 胰蛋白酶-EDTA溶液(NACALAI TESQUE,目录号:32778-34)
  39. 氢氧化钠(NaOH)(和光纯药工业,目录号:198-13765)
  40. 氯化铵(NH4Cl)(Wako Pure Chemical Industries,目录号:017-02995)
  41. 碳酸氢钾(KHCO 3)(Wako Pure Chemical Industries,目录号:166-03275)
  42. 鼠干细胞因子(SCF)(PeproTech,目录号:250-03)
  43. 鼠白细胞介素-3(IL-3)(PeproTech,目录号:213-13)
  44. 鼠白细胞介素-6(IL-6)(PeproTech,目录号:216-16)
  45. 鼠粒细胞巨噬细胞集落刺激因子(GM-CSF)(PeproTech,目录号:315-03)
  46. 牛血清白蛋白(BSA)(Wako Pure Chemical Industries,目录号:019-23293)
  47. IMDM粉末(Thermo Fisher Scientific,Gibco TM,目录号:12200036)
  48. 碳酸氢钠(NaHCO 3)(Wako Pure Chemical Industries,目录号:195-14515)
  49. 甲基纤维素(粘度:4000cP)(Sigma-Aldrich,目录号:M0512)
  50. 针对鼠类Hoxa9和Gapdh的TaqMan探针探针
    Hoxa9(Thermo Fisher Scientific,Applied Biosystems TM,目录号:Mm00439364_m1)
    (Gapdh)(Thermo Fisher Scientific,Applied Biosystems TM,目录号:Mm99999915_g1)
  51. TaqMan快速高级主混合物(Thermo Fisher Scientific,Applied Biosystems TM,产品目录号:4444557)
  52. 25X磷酸盐缓冲盐水Ca2 + / Mg2 + / PBS(见食谱)
  53. D10媒体(见食谱)
  54. R10媒体(见食谱)
  55. R10W10媒体(见食谱)
  56. 0.5M EDTA溶液(见食谱)
  57. ACK裂解缓冲液(见食谱)
  58. SM缓冲区(见食谱)
  59. 细胞因子库存(见食谱)
  60. 交流媒体(见食谱)

设备

  1. 5%CO 2培养箱37℃
  2. 5%CO 2培养箱32°C
  3. MACS多中心(Miltenyi Biotec,目录号:130-042-303)
  4. 移液器P2,P20,P200,P1000
  5. Portable Pipet-Aid XP移液管控制器(Drummond Scientific,目录号:4-000-101)
  6. 显微镜
  7. 细胞计数器
  8. 高压灭菌器
  9. 1升玻璃瓶
  10. 摇床
  11. 手术剪刀和钳子
  12. 层流柜
  13. 离心15毫升锥形管
  14. MiniMACS分离器(Miltenyi Biotec,目录号:130-042-102)
  15. StepOnePlus TM实时PCR系统(Thermo Fisher Scientific,Applied Biosystems TM,型号:StepOnePlus TM,目录号:4376600)。
  16. 用于Western印迹和SDS-PAGE的通用设备

软件

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

程序

附表(见图1):
第一天1.开始从冷冻的原料培养PLAT-E细胞 第3天。Replate PLAT-E细胞转染
第4天。转染PLAT-E细胞
第5天c-kit阳性细胞的制备
第6天。将逆转录病毒转导到c-kit阳性细胞中
第7天加入G418
第十二天第一段
第十七天第二次通过
第22天:殖民统计和第三次通过
第27天殖民地计数


图1.小鼠骨髓祖细胞转化实验的时间表

<强>

  1. 病毒制备
    1. 在第1天,从冷冻原液中解冻PLAT-E细胞,并在37℃5%CO 2中用10ml D10培养基(见食谱)在10cm培养皿中培养4×10 6个细胞。 2>孵化器。
    2. 在第3天,在37℃的5%CO 2中用5ml D10培养基重新铺板6cm胶原蛋白包被的培养皿中的8×10 5 PLAT-E细胞,孵育器(一个阳性对照[MLL-ENL],一个阴性对照[空载体; pMSCV-neo],两个用于G418选择的模拟对照和样品[至多16个样品])。
    3. 第4天,用8μgDNA和20μlLipofectamine TM 2000转染试剂转染PLAT-E细胞。转染时,细胞汇合度应为60-70%。 (详细地说,在500μlOpti-MEM TM培养基中稀释8μgDNA,并在500μlOpti-MEM TM培养基中稀释20μlLipofectamine TM 2000转染试剂, 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,加入DNA-Lipofectamine混合物每个培养皿中,并在37℃,5%CO 2培养箱中孵育。)6小时后,用新鲜的D10培养基代替培养基并在32℃5%CO 2,孵化2天。
      注意:病毒上清液应该在每个实验中新鲜制备,因为逆转录病毒是不稳定的储存。

  2. 制备c-kit(CD117)阳性的祖细胞
    1. 在第5天,从五周龄的C57BL / 6J小鼠(图2A)尽可能彻底地收获股骨和胫骨并去除周围肌肉。
    2. 切割股骨和胫骨的末端,并用10毫升PBS冲洗骨髓(参见食谱),使用附带21 G针的10毫升注射器(图2B)(视频1)。

      视频1


    3. 通过21G针轻轻匀化骨髓细胞几次
    4. 在室温下将细胞在400×g下旋转5分钟(图2C左),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),在室温下将细胞在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(参见食谱)中,并在室温下400×g下旋转5分钟(图2C右),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 SM缓冲液并在室温下400×g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. 在磁力架上安装MS柱,并用1 ml SM缓冲液平衡。
    12. 将预分离过滤器放在色谱柱顶部,通过预分离过滤器(图2D)将细胞加载到MS色谱柱上。


      图2. c-kit阳性祖细胞分离的代表性图像A.收获的胫骨和股骨; B.用PBS冲洗骨髓; C.通过ACK缓冲液处理去除红细胞; D.通过MACS的c-kit阳性细胞分离。


    13. 用1 ml SM缓冲液清洗柱子两次
    14. 从磁力架上取下柱子,放在新的15毫升锥形管上。
    15. 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。
    16. 加入10ml R10W10培养基,室温下400×g离心5min,取出上清液。
    17. 重悬在1毫升R10W10中的所有细胞,并转移到48孔板的一个孔中。
    18. 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. 病毒转导和细胞培养
    1. 在第6天,计数细胞。预计每只小鼠总共有1-2×10 6个c-kit阳性细胞。
    2. 加入适量的R10W10培养基以制备1×10 5个细胞/ ml悬浮液并加入1/250体积的10mg / ml聚凝胺溶液。
    3. 将0.5 ml细胞悬液分装到15 ml锥形管中(另外准备两个模拟感染对照)。
    4. 用5毫升注射器吸取5毫升病毒上清液,将一个过滤器(0.45微米)连接到5毫升注射器,并将病毒上清直接添加到c-kit阳性细胞悬液。

    5. 在32℃下将细胞悬浮液在1,100×g下旋转2.5小时(Spinoculation)。
    6. 在接种过程中,在PLAT-E细胞中加入1ml PBS,使用细胞提取器收获细胞,并将细胞转移到新管中。
    7. 再次用1ml PBS洗涤包装细胞,并在500μlWB裂解缓冲液中制备全细胞裂解液。
    8. 接种后,取出病毒上清液(留下大约200μl培养基),将细胞重新悬浮于残留培养基中,加1ml AC培养基(参见食谱),使用装有18G针头的1ml注射器,通过涡旋混合。
      注意:使用1毫升注射器和针头取1毫升AC介质,因为这种介质非常粘稠。移液器不能使用。
    9. 将含有细胞的15-ml锥形管在37℃5%CO 2培养箱中培养过夜。
    10. 第7天,加入20μlG418溶液,涡旋混合,并使用装有18G针的1ml注射器将细胞转移至12孔板中。对于两个模拟控制,在一个管中添加G418以确保G418选择正常。
      注意:样品应放置在12孔板的中央6个孔中,PBS应添加到外围6个孔中以防止AC介质中的培养物干燥。
    11. 在37℃5%CO 2培养箱中孵育5天(图3)。如果培养过程中由于细胞过多而使培养基变成橙色,则添加1毫升含G418的AC培养基。
      注:保持良好的文化条件是重要的。过度生长会严重影响下一轮文化中的殖民地形成。
    12. 任选地,可以在第一轮培养期间通过WB检查包装细胞中转基因的蛋白质表达。


      图3.第一轮和第四轮培养期间的菌落图像

  4. 第一段
    1. 在第12天,用10ml PBS收集细胞,转移到15ml锥形管中,并在室温下400×g下旋转5分钟。
    2. 用10毫升的PBS洗细胞。
      注意:通过剧烈的移液来清除AC介质的碎屑是非常重要的。
    3. 将细胞重悬于1ml R10W10培养基中,并计数细胞。
    4. 转移4×10 4细胞到一个新的15毫升锥形管,并添加适量的R10W10准备200-μl细胞悬液。
    5. 加入1毫升AC介质,涡旋混合。 使用1-ml注射器连接18 G针将细胞转移到新的12孔板的孔中。
      注意:样品应放置在12孔板的中央6个孔中,PBS应添加到外围6个孔中以防止AC介质中的培养物干燥。
    6. 将细胞在37℃5%CO 2培养箱中孵育5天。如果在培养过程中媒体变成橙色,则再向孔中添加1毫升交流媒质。
    7. 任选地,如方法E中所述,将残余细胞收集到新管中并裂解含有RNeasy Mini试剂盒的1%β-巯基乙醇的RLT裂解缓冲液中的细胞用于RNA分离。

  5. 细胞收集和分析
    1. 使用RNeasy®mini试剂盒分离RNA,并使用带有oligo-dT引物的Superscript®III第一链合成系统合成cDNA。
    2. 通过定量PCR评估基因表达。在通过MLL-融合基因进行的骨髓祖细胞转化的情况下,Hoxa9基因表达是永久化的一个很好的指标。

  6. 第二段

    1. 第17天,收集细胞并计数
    2. 培养2×10 4个细胞/孔。

  7. 殖民统计和第三次通过
    1. 在第22天,在显微镜下计数菌落数。
      注意:在载体对照中不应该观察到菌落。设定一个定义殖民地的标准并在整个研究中使用这个定义是很重要的。我们通常认为一个有100多个细胞群作为殖民地。
    2. 像第一代一样收获和计数细胞。
    3. 培养1×10 4个细胞/孔。

  8. 菌落计数
    在第27天,在显微镜下计算菌落数(图3)。

数据分析

  1. 由于实验的变化,这个检测必须执行至少三次。
  2. 为了统计分析,我们使用GraphPad Prism软件。为了比较两个数据集,我们执行Student's t -test。为了比较三个或更多的数据集,我们进行方差的单向分析和事后Tukey校正。

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. 培养WE10在R10媒体融合
    2. 当媒体变成橙色时,收集和过滤媒体
      (0.22-μm)
    3. 分装和存储在-80°C
    4. 加入55毫升FBS,55毫升WEHI-3培养基和5.5毫升P / S溶液到500毫升RPMI 1640中。
  5. 0.5M EDTA溶液
    1. 称取93.06克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保存
  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. 在PBS + 0.1%BSA中将细胞因子(SCF,IL-3,IL6,GM-CSF)溶解至50μg/ ml
    2. 分装和存储在-80°C
  9. AC介质(或者STEMCELL Technologies,Vancouver,Canada的Methocult M3231)
    1. 将IMDM粉末溶于500ml蒸馏水中,加入3g NaHCO 3和过滤器(0.2-μm)。
    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. 将无菌甲基纤维素溶于300ml无菌水和500ml IMDM的振荡器中过夜。
    5. 加200毫升的FBS和7微升的β-巯基乙醇
    6. 分装每瓶100毫升,并储存在-20°C
    7. 使用前,加20μLSCF,20μLIL-3,20μLGM-CSF和1mlP / S溶液到100ml培养基中

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等人的先前报道(1997)。作者宣称没有利益冲突。

参考

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引用:Okuda, H. and Yokoyama, A. (2017). Myeloid Progenitor Transformation Assay. Bio-protocol 7(23): e2626. DOI: 10.21769/BioProtoc.2626.
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