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Mar 2019
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Modeling NOTCH1 driven T-cell Acute Lymphoblastic Leukemia in Mice
NOTCH1诱导小鼠T细胞急性淋巴细胞白血病模型的建立   

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Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that arises from transformation of T-cell primed hematopoietic progenitors. Although T-ALL is a heterogenous and molecularly complex disease, more than 65% of T-ALL patients carry activating mutations in the NOTCH1 gene. The majority of T-ALL–associated NOTCH1 mutations either disrupt the negative regulatory region, allowing signal activation in the absence of ligand binding, or result in truncation of the C-terminal PEST domain involved in the termination of NOTCH1 signaling by proteasomal degradation. To date, retroviral transduction models have relied heavily on the overexpression of aggressively truncated variants of NOTCH1 (such as ICN1 or ΔE-NOTCH1), which result in supraphysiological levels of signaling activity and are rarely found in human T-ALL. The current protocol describes the method for mouse bone marrow isolation, hematopoietic stem and progenitor cell (HSC) enrichment, followed by retroviral transduction with an oncogenic mutant form of the NOTCH1 receptor (NOTCH1-L1601P-ΔP) that closely resembles the gain-of-function mutations most commonly found in patient samples. A hallmark of this forced expression of constitutively active NOTCH1 is a transient wave of extrathymic immature T-cell development, which precedes oncogenic transformation to T-ALL. Furthermore, this approach models leukemic transformation and progression in vivo by allowing for crosstalk between leukemia cells and the microenvironment, an aspect unaccounted for in cell-line based in vitro studies. Thus, the HSC transduction and transplantation model more faithfully recapitulates development of the human disease, providing a highly comprehensive and versatile tool for further in vivo and ex vivo functional studies.

Keywords: Mouse model (小鼠模型), In vivo (体内), T-ALL (T细胞急性淋巴细胞白血病), NOTCH1 signaling (NOTCH1 信号), NOTCH1 mutations (NOTCH1 突变), Leukemia development (白血病形成), Retrovirus (逆转录酶病毒), Transformation (转化), Hematopoietic stem cell (造血干细胞), Bone marrow (骨髓), Transplantation (移植)

Background

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological tumor arising from the malignant transformation of hematopoietic progenitor cells primed towards T-cell development. T-ALL accounts for up to 15% of pediatric and 25% of adult ALL (acute lymphoblastic leukemia) and occurs more frequently in males than females (Goldberg et al., 2003). Despite being characterized by a high level of heterogeneity and molecular complexity (Liu et al., 2017), more than 65% of T-ALL patients carry gain-of-function mutations in the NOTCH1 gene (Weng et al., 2004). The resulting constitutive activation of NOTCH1 signaling is therefore the most prominent oncogenic pathway in T-cell transformation.

NOTCH1 proteins are developmentally conserved type I transmembrane receptors that play a prominent instructive role in T-cell lineage commitment and cell growth and proliferation during thymocyte development (Radtke et al., 1999; Defto and Bevan, 2000). NOTCH1 signaling is initiated upon binding of transmembrane ligands expressed on the surface of neighboring cells. This interaction prompts cleavage of the extracellular domain of the receptor by the ADAM10 metalloprotease followed by γ-secretase cleavage in the transmembrane domain of the receptor. Subsequent release from the membrane results in translocation of the intracellular domain (ICN1) to the nucleus and transcriptional activation of target genes (Kopan and Ilagan, 2009; Andersson et al., 2011). Finally, termination of NOTCH1 signaling is regulated by phosphorylation of the C-terminal PEST domain, which targets ICN1 for ubiquitination and FBXW7-mediated proteasomal degradation (O'Neil et al., 2007; Thompson et al., 2007). Constitutive activation of NOTCH1 in human T-ALL results from disruption of the mechanisms stringently regulating this multi-step process. The majority of T-ALL-associated mutations result either in disruption of the negative regulatory region, resulting in NOTCH1-signaling activation in the absence of ligand binding, or truncation of the C-terminal PEST domain which allows ICN1 to evade proteasomal degradation, thus impairing termination of signaling activity (Weng et al., 2004). In the past decade, the introduction of intensive combination chemotherapy approaches has increased the cure rate of pediatric T-ALL to nearly 90%. Nonetheless, the prognosis for patients with primary refractory or relapse T-ALL remains very poor (Goldberg et al., 2003; Litzow and Ferrando, 2015), underscoring the need to further decipher the molecular pathology underlying T-ALL transformation, identification of more specific therapeutic targets, and development of more effective and less toxic multi-drug therapies.

To date, in vitro human T-ALL-derived cell-line cultures remain the most commonly employed experimental approach to the study of T-ALL. Notably, this model does not correct for genetic drift associated with decade-long periods of culture and fails to account for the crosstalk between leukemia cells and the tumor microenvironment (Passaro et al., 2015; Pitt et al., 2015). Animal models therefore provide an alternative approach to functional studies that more accurately recapitulate human T-ALL in vivo. The mouse hematopoietic stem cell (HSC) retroviral transduction and transplantation model has been extensively applied to mimic initiation and progression of NOTCH1-driven T-ALL (Wendorff et al., 2010; Medyouf et al., 2011; Gachet et al., 2013). It has been shown that retroviral expression of ICN1 or ΔE-NOTCH1 (the extracellular domain-truncated form of the receptor) in murine bone marrow progenitors drives thymus-independent T-cell development and rapidly induces T-ALL development (Pear et al., 1996; Perez-Garcia et al., 2013). However, it should be noted that these aggressively truncated variants of NOTCH1, which result in supraphysiological levels of signaling activity, are rarely found in human T-ALL (Chiang et al., 2008). In fact, ΔE-NOTCH1 truncated alleles are expressed in only a small subset of patients carrying chromosomal translocations (1%) (Ellisen et al., 1991; Palomero et al., 2006). Much more commonly, human T-ALLs harbor acquired gain-of-function point mutations, which occur predominantly within the extracellular HD and the C-terminal PEST domains (Weng et al., 2004). Of note, 20% of T-ALL patients exhibit co-occurrence of these two mutation-driven activating mechanisms, which generate levels of activity well below those of ΔE-NOTCH1 (Chiang et al., 2008). As the outcome of NOTCH signaling is highly dose dependent in many developmental settings (Artavanis-Tsakonas et al., 1999; Bray, 2016), we hypothesized that the level of activity has a similar impact on the process of leukemogenesis. The current protocol describes the method for mouse hematopoietic stem cell (HSC) retroviral transduction with an oncogenic mutant NOTCH1 receptor (NOTCH1-L1601P-ΔP) that closely resembles the gain-of-function mutations found in patient samples. The L1601P-ΔP mutant contains a common HD point mutation (L1601P) that enables ligand-independent activation (Malecki et al., 2006), together with a frameshift mutation that results in truncation of amino acids 2,473-2,555, which comprise the negative regulatory PEST domain (ΔP). We outline the method for isolation of bone marrow cells from mice, enrichment for hematopoietic stem and progenitor cells, followed by retroviral transduction and transplantation into isogenic recipients (Figure 1). In this model, forced expression of constitutively active mutant NOTCH1 typically results in a wave of extrathymic T-cell lymphopoiesis 3 weeks after transplantation, which manifests as the transient appearance of GFP+ preleukemic CD4+ CD8+ double-positive (DP) immature T cells in peripheral blood (Figure 4). Transformation resulting in lethal leukemia progression occurs 12-15 weeks following transplantation (Wendorff et al., 2019) (Figure 4). When combined with constitutive- or inducible-knockout transgenic models, the protocol can be easily modified to address the role of candidate genes in NOTCH1-driven T-ALL transformation, tumor initiation and tumor progression (Wendorff et al., 2019). Furthermore, this approach provides a rapid and efficient model system for pre-clinical assessment of potential novel anti-leukemic drugs (Herranz et al., 2015; Sanchez-Martin et al., 2017).

Materials and Reagents

  1. Pipette tips
  2. Parafilm
  3. Kimwipes Delicate Task Wipers, 1-Ply (Kimberly-Clark Professional, catalog number: 06-666 )
  4. 150 mm TC-treated cell culture dishes (Corning, Falcon, catalog number: 353025 )
  5. 24-well TC-treated microplates (Corning, Falcon, catalog number: 353847 )
  6. 5 ml round-bottom polystyrene tubes (Corning, Falcon, catalog number: 352008 )
  7. 15 ml Conical bottom Centrifuge Tubes (Corning, Falcon, catalog number: 352196 )
  8. 50 ml Conical bottom Centrifuge Tubes (Corning, Falcon, catalog number: 352070 )
  9. 3 ml BD Luer-Lok tip Syringes (Becton Dickinson, catalog number: 309657 )
  10. 30 ml BD Luer-Lok tip Syringes (Becton Dickinson, catalog number: 302832 )
  11. Sterile 25 mm PES syringe filters, 0.45 µm pore size (Thermo Fischer Scientific, catalog number: 7252545 )
  12. 40 µm cell strainer (Corning, Falcon, catalog number: 352340 )
  13. 70 µm cell strainer (Thermo Fisher Scientific, catalog number: 22-363-548 )
  14. Amicon Ultra-15 Centrifugal 15 ml sample volume filter units NMWL 100KDa (EMD Millipore, catalog number: UFC910008 )
  15. U-100 Insulin syringes, 1 ml, 26 G (BD Biosciences, catalog number: 329652 )
  16. U-100 Insulin syringes, 0.5 ml, 29 G½ (Exelint international, catalog number: 26028 )
  17. Goldenrod Animal Lancet, 5 mm (Medipoint, catalog number: NC9891620 )
  18. LS Columns for negative cell selection (Miltenyi Biotec, catalog number: 130-042-401 )
  19. C57BL/6 bone marrow donor mice aged 6-12 weeks and isogenic C57BL/6 bone marrow recipient mice (min. 6 weeks of age) (The Jackson Laboratory, catalog number: 000 664 )
  20. 293T cells (ATCC, catalog number: CRL-3216 )
  21. pCL-Eco Retrovirus Packaging Vector (Addgene, catalog number: 12371 ) (Naviaux et al., 1996)
  22. pMSCV-NOTCH1-L1601P-ΔP (Chiang et al., 2008)
  23. jetPEI DNA Transfection Reagent provided with 150 mM NaCl solution (Polyplus, catalog number: 101-10N )
  24. PBS: Cell Culture Phosphate Buffered Saline (1x) without calcium and magnesium (Corning, catalog number: 21040CV )
  25. DMEM (Corning, catalog number: 10-013-CV )
  26. Opti-MEM I Reduced Serum Medium, GlutaMAX Supplement (Gibco, catalog number: 51985 )
  27. Fetal Bovine Serum (FBS), heat inactivated and sterile (Sigma-Aldrich, catalog number: F4135 )
  28. Penicillin (10,000 IU) and Streptomycin (10,000 µg/ml) in a 100-fold working concentration (Corning, catalog number: 30002CI )
  29. HEPES 1 M (Gibco, catalog number: 15630080 )
  30. 0.5 M EDTA (Lonza, catalog number 51201 )
  31. β-mercaptoethanol, 55 mM (Gibco, catalog number: 21985023 )
  32. Trypan blue (Gibco, catalog number: 15250061 )
  33. ACK Lysing Buffer (Quality Biologicals, catalog number: 118-156-721 )
  34. CD117 Microbeads, mouse (Miltenyi Biotec, catalog number: 130-091-224 )
  35. Lineage Cell Depletion Kit, mouse (Miltenyi Biotec, catalog number: 130-090-858 )
  36. Polybrene Infection/Transfection Reagent (EMD Millipore, catalog number: TR-1003-G )
  37. CountBright Absolute Counting Beads for flow cytometry (Invitrogen, catalog number: C36950 )
  38. Recombinant murine IL-3 (Peprotech, catalog number: 213-13 )
  39. Recombinant murine IL-6 (Peprotech, catalog number: 216-16 )
  40. Recombinant murine IL-7 (Peprotech, catalog number: 217-17 )
  41. Recombinant murine Flt3-L (Peprotech, catalog number: 250-31L )
  42. Recombinant murine SCF (Peprotech, catalog number: 250-03 )
  43. Recombinant murine TPO (Peprotech, catalog number: 315-14 )
  44. Anti-mouse Sca-1 (Ly-6A/E) monoclonal antibody (clone D7), PE-Cyanine7 conjugate (eBioscience, catalog number: 25-5981 )
  45. (Optional) Anti-mouse CD4 monoclonal antibody (clone RM4-5), APC-conjugate (BD Biosciences, catalog number: 561091 )
  46. (Optional) Anti-mouse CD8 monoclonal antibody (clone 53-6.7), PE-Cyanine7 conjugate (BD Biosciences, catalog number: 561097 )
  47. DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) (Invitrogen, catalog number: D3571 )
  48. Heparin, 1,000 USP units/ml (Sagent Pharmaceuticals, catalog number: 25021-400-30 )
  49. Ketamine HCl injection (100 mg/ml: controlled substance); consult institution’s veterinarian
  50. Xylazine sterile solution (20 mg/ml: AKORN Animal Health; catalog number: 139-236 ); consult institution’s veterinarian
  51. Staining medium (SM; see Recipes)
  52. 293T culture/transfection medium (see Recipes)
  53. Growth-factor supplemented transduction medium (see Recipes)
  54. Ketamine/xylazine anesthesia cocktail (see Recipes)

Equipment

  1. 1 ml/200 µl/20 µl pipettes
  2. Mortar and pestle
  3. A cell culture incubator, 37 °C/5% CO2
  4. Dissection tools: scissors, forceps, scalpel blade (recommended source: Fine Science Tools www.finescience.com)
  5. Hemocytometer (recommended) or automated cell counter
  6. Laminar flow biosafety cabinet
  7. Refrigerated centrifuge fitted with a swing-bucket rotor and plate adapters
  8. MACS MultiStand (Miltenyi Biotec, catalog number: 130-042-303 )
  9. MidiMACS Separator (Miltenyi Biotec, catalog number: 130-042-302 )
  10. A Fluorescence Microscope with green fluorescent protein (GFP) excitation/emission filters
  11. A Flow Cytometer instrument with a Blue (488 nm), Violet (405 nm), Green (561 nm) and Red (633 nm) laser (recommended: LSRFortessa, BD Biosciences)
  12. A gamma-source or alternative small-animal irradiator

Software

  1. FlowJo (FlowJo LLC, Ashland, OR, USA)
  2. GraphPad Prism Software (La Jolla California USA, www.graphpad.com)

Procedure

This approach to retroviral transduction and bone marrow transplantation can be used to overexpress any oncogene of interest to generate either lymphoid or myeloid leukemia in mice. Transformation efficiency, kinetics of disease progression and tumor penetrance will depend on both the tumorigenicity of the oncogene used and experimental variables, such as the number of transplanted transduced hematopoietic stem cells.
Note: Please refer to the Notes section of this protocol for additional information and see Figure 4 for a comparison of the pMSCV-NOTCH1-L1601P-ΔP and ΔE-NOTCH1 leukemia models (recommended cell numbers, kinetics of leukemia development and penetrance).

The procedure described here has been optimized for modeling T-ALL development driven specifically by the L1601P-ΔP mutant form of the NOTCH1 receptor. For example, transfection of 293T cells is modified to account for reduced transfection efficiency when using larger constructs, such as the pMSCV-NOTCH1-L1601P-ΔP-ires-GFP (> 15 kb) and counteract the resulting reduction in viral titer. Furthermore, the protocol recommends bone marrow isolation from the hip bones and spinal vertebrae, in addition to the tibiae and femurs. This approach greatly increases the total number of bone marrow stem and progenitor cells available for subsequent transduction, while simultaneously reducing the number of experimental animals needed. Although all mice listed in this protocol (both donor and recipient mice) are on the C57BL/6 background, other mouse strains may be used if bone marrow donor and recipient mice are on congenic genetic backgrounds. However, it is important to remember that each strain has unique background alleles that may interact with and modify the expression of a mutation, transgene or other genetic insert (https://www.jax.org/news-and-insights/2006/june/the-importance-of-genetic-background-in-mouse-based-biomedical-research).

Please note that the experimental procedure outlined in this protocol requires 6 consecutive days of hands-on activity (Days 0-5), with a subsequent follow-up on Day 21 (Figures 1A and 1B; Figure 4).

Important: All experimental procedures must be approved by the local animal care committee.
Notes:

  1. Prior to beginning the protocol, calculate the number of bone marrow donor mice needed based on the desired number of recipient animals; as a “rule of thumb”, when harvesting marrow from only the 2 hind legs, n = 1 bone marrow donor is sufficient for transplantation into n = 2-3 recipient animals; when harvesting marrow from all bones (hind and fore limbs, pelvic bone and spinal column), n = 1 bone marrow donor is sufficient for transplantation into n = 6-8 recipient animals.
  2. An additional untreated congenic wild-type mouse is needed as a source of helper bone-marrow cells on Day 5 of the protocol.


    Figure 1. Experimental design for generation of oncogenic NOTCH1-driven T-ALL from mouse hematopoietic stem cells. A. Cartoon representation of key experimental steps. B. Timeline of the experimental strategy.

Transient transfection of 293T cells
Day 0:

Plate 293T cells 24-48 h before transformation at a density that will result in approximately 60-70% confluence at time of transfection (for 293T medium; see Recipes). Use 293T cells that have been passaged no less than 2x after thawing of a fresh vial of live-frozen cells. Do not exceed a total of 20 passages. Use only tested mycoplasma negative cells (Young et al., 2010).
Note: Plating density should be optimized based on the size of the culture plate and the growth kinetics of the 293T cells being used for virus production. The values below have been optimized for 150 mm tissue culture plates, with a conservative ratio of 1:1 of 293T culture plates to the number of donor mice and total bones used for bone marrow isolation.

Day 1:

In the evening, perform transient transfection of 293T cells (Table 1); see https://www.polyplus-transfection.com/wp-content/uploads/2015/09/CPT_101_jetPEI_vU.pdf for general guidelines.

  1. Prepare the DNA and transfection mix in separate tubes in sterile conditions:

    Table 1. Amount of transfection reagent and retroviral construct recommended per 1x 150 mm plate of 293T cells (scale up as needed)


  2. Add the contents of Tube #1 to Tube #2 (always add transfection reagent to DNA mix and not vice versa), vortex gently and allow to stand at room temperature for 20-30 min.
  3. Add the mix to 293T cells drop-wise; 1 ml per 150 mm plate.
  4. Incubate overnight (12-16 h).

Day 2:

  1. Transient transfection of 293T cells, continued
    In the morning, remove transfection medium and very gently replace with 13-15 ml fresh 293T medium. Place in incubator until the next day.
    Notes:
    1. 293T cells will begin to produce GFP within 12 h of transfection. To confirm successful transfection and obtain a qualitative estimate of transfection efficiency, it is recommended to verify GFP production using a fluorescence microscope 16-24 h after transfection; transfected virus-producing cells with appear brightly fluorescent.
    2. Perform all media changes with extreme care as cells have been sensitized by the transfection reagent and detach from the plate more easily with continued culture.
    3. From this point onwards observe all safety requirements when handling BSL-2 hazardous materials.

  2. Isolation of bone marrow cells and CD117+ cell enrichment
    Notes:
    1. This protocol recommends bone marrow isolation by crushing of bones using a mortar and pestle, an approach that results in higher and more consistent cell yields, while maintaining high cell viability. While isolation of bone marrow from hip bones and spinal vertebrae appears more cumbersome to novice researchers, it greatly increases the cell yield per mouse and decreases the number of experimental animals required for bone marrow extraction, a pivotal consideration when using transgenic mouse models. While dissection of tibiae and femurs from experimental mice is well established in the field, obtaining the hip bones and spinal vertebrae is less common. We highly recommend viewing the video tutorial published by Lo Celso and Scadden (2007) prior to undertaking this approach.
    2. Retroviral transduction of mouse bone marrow cells is most commonly performed using negative selection of total lineage-negative bone marrow cells. Positive selection of CD117+ cells, as described here, tips the scales towards transduction of hematopoietic stem cells, which results in increased transformation efficiency of the target population with self-renewal potential.
    3. Keep staining medium (SM) and the bone marrow cell suspension (BM) on ice at all times. Perform all centrifugation steps at 4 °C.
    4. The values provided below are based on total BM isolated from all bones of n = 1 adult mouse (approx. 8-10 weeks of age) and should be scaled up appropriately based on the target number of bone marrow donor animals.

    1. Sacrifice donor mice: CO2 asphyxiation followed by cervical dislocation is recommended.
    2. Dissect the tibia, femurs and pelvic bones from each donor mouse; the total expected bone-marrow cell yield is approximately 120 x 106/animal, but varies with age of the donor animals. If needed, the total cell yield can be increased to 160 x 106-200 x 106/animal by additionally isolating the spinal vertebrae.
    3. Clean the bones by scraping off muscle tissue using a scalpel blade. Remove remaining tissue by rubbing the bones between your fingers using Kimwipes/paper towel. It is recommended to gently break off the cartilage of the kneecaps and other joints by dislocation rather than cutting the bone, as this preserves the integrity of the bone, results in higher yields and reduces the risk of contamination. If isolating spinal vertebrae, separate the vertebrae into 3-4 individual segments and remove the spinal cord. Clean thoroughly by rubbing with Kimwipes. Keep cleaned bones in cold SM on ice.
    4. Although not absolutely necessary, it is recommended to perform all subsequent steps in sterile conditions in a laminar flow biosafety cabinet to prevent contamination. Crush the bones in 5 ml cold SM using a mortar and pestle (crush, rather than grind, to retain high cell viability). Repeatedly aspirate and eject the cell suspension using a 3 ml syringe to generate a single-cell suspension and pass through a 40 µm cell strainer into a 50 ml collection tube placed on ice. Add 5 ml SM to the mortar and repeat this sequence approximately 3 times, until the bones are thoroughly crushed and appear translucent/white in color.
    5. After the final round of crushing, wash the strainer with 5 ml SM; this results in approximately 40 ml of single-cell bone-marrow suspension per donor mouse.
    6. Pellet cells at 500 rcf at 4 °C for 7 min.
    7. Aspirate/pour off the SM (the pellet will appear very RED).
    8. Perform red blood cell lysis by resuspending the pellet in 5 ml ACK buffer and incubate at room temperature for 5 min. Pass the cell suspension through a 70 µm cell strainer into a 50 ml conical tube placed on ice containing at least 20 ml SM to stop the lysis reaction, then fill the tube to capacity with SM to further dilute the ACK lysis buffer.
    9. Pellet cells at 400 rcf at 4 °C for 10 min.
    10. Aspirate/pour off the supernatant (the pellet should now appear near WHITE) and carefully resuspend the cell pellet in 5 ml cold SM. Add 15 ml cold SM and gently pulse-vortex to obtain 20 ml of a uniform cell suspension.
    11. Determine the number of viable cells (Trypan blue exclusion) either by manual cell counting using a hemocytometer or an automated cell counter.
      Note: Due to the heterogeneity of total bone marrow cells, the former approach results in more accurate quantification of cell number.
    12. Continue with CD117+ positive-cell selection using anti-CD117 Microbeads following the manufacturer’s instructions (https://www.miltenyibiotec.com/US-en/resources/technical-documents/data-sheets.html). In the final step, elute into a 15 ml Eppendorf tube and add 10 ml SM. Pellet cells at 4 °C for 5 min at 500 rcf.
    13. Resuspend the CD117+ cell-enriched pellet in 2 ml cold SM and proceed with cell quantification. Keep cells on ice.
    14. Manual cell counting using a hemocytometer and Trypan blue exclusion of dead cells is recommended at this step of the protocol to ensure accurate cell counts. When harvesting cells from ALL bones (as above), the total expected yield is approximately 5 x 106 CD117+ cells/donor animal.
    15. Plate CD117+-enriched cells in a 24-well tissue culture plate in 2 ml/well growth-factor supplemented medium (for GF medium, see Recipes) at a density of 0.5 x 106 cells/ml and culture overnight.

Day 3: First round of virus collection and bone-marrow progenitor cell transduction


  1. First collection and concentration of the viral supernatant from 293T cultures
    Note: Perform the following steps at room temperature in a Level 2 biosafety cabinet and observe all safety requirements of handling BSL-2 hazardous materials.
    1. Collection and filtration the viral supernatant:
      1. Collect the viral supernatant (VSN) from 293T cultures and remove large cell debris by passing through a 70 µm strainer into a 50 ml conical collection tube.
      2. Carefully replenish cultures with 13-15 ml fresh 293T medium for the second collection of VSN on Day 4.
      3. Remove small debris and aggregates by passing the VSN through a 0.45 µm PES syringe filter (this is most easily performed by aspirating the VSN from the 50 ml conical collection tube into a 30-60 ml syringe fitted with a large 18 G½ gauge needle, turning the syringe upwards to prevent spillage and contamination, and replacing the needle with a syringe filter prior to ejecting the VSN into a fresh 50 ml conical-bottom collection tube).
      Note: Two consecutive filtration steps remove all particles that may result in clogging of the Amicon Ultra concentration column in the next step.
    2. Concentration of the viral supernatant
      1. Transfer VSN into an Amicon filtration column tube to maximum capacity (Amicon Ultra = 15 ml).
      2. Centrifuge at 2,500 rcf at room temperature until approximately 80% of the VSN has passed through the filtration unit (typical final concentrate volume/15 ml VSN volume = 200 µl). If VSN is collected from > 1 x 150 mm 293T virus-producing dish, repeat Steps A2a-A2b using the same filtration unit until the desired final volume of VSN is reached. Transfer the final volume of concentrated VSN to an Eppendorf tube. Discard the Amicon filtration unit.
      Note: Generate 100 µl of VSN (20x concentration) for each well of the 24-well culture plate containing CD117+-enriched bone marrow in a volume of 2 ml. i.e., if CD117+-enriched bone marrow cells were plated in 12 wells on Day 2 of the protocol, concentrate the VSN to a final volume of 1,200 µl. Adjust volume as needed with fresh OptiMEM cell culture medium. Remember to add the appropriate amount of Polybrene (Step B1 below).

  2. First transduction of CD117+-enriched bone marrow progenitor cells
    Note: Pre-warm the centrifuge for spin-infection to 28 °C. Set the deceleration speed (break) to 0.
    1. Add Polybrene reagent to the Eppendorf tube containing concentrated VSN to a final concentration of 80 µg/ml (20x).
    2. Add 100 µl of the 20x VSN containing 20x Polybrene to each well of the 24-well plate containing CD117+-enriched bone marrow cells in 2 ml GF-supplemented media.
    3. Seal the plate with parafilm to prevent contamination of the centrifuge during spin-infection.
    4. Centrifuge at 1,100 rcf at 28 °C for 90 min.
    5. Carefully remove parafilm and return the plate to a TC incubator. Allow the cells to recover for at least 6 h.
    6. After 6-8 h, gently remove ½ of the media, ~1 ml (gently tilt the plate; use a 1 ml pipettor and keep the pipette tip level with the surface of the media while aspirating to prevent aspiration of bone marrow cells).
    7. Add 1 ml of fresh GF-supplemented transduction medium and allow cells to recover overnight (12-16 h).

Day 4: Second round of virus collection and bone-marrow progenitor cell transduction


  1. Second collection and concentration of the viral supernatant from 293T cultures
    Repeat Steps A1a-A1c and A2a-A2b as on Day 3 of the protocol.

  2. Second transduction of CD117+-enriched bone marrow progenitor cells, previously transduced on Day 3 and cultured overnight in GF-supplemented medium.
    Repeat Steps B1-B7 as on Day 3 of the protocol.

  3. Irradiation of recipient mice
    Notes:
    1. It is recommended to use 8-week-old mice as recipients, although a 6-12-week range is acceptable. As recipient mice should be irradiated at least 8 h prior to transplantation, this protocol recommends irradiation on the evening of the day before transplantation. Deliver a total body dose (TBI) of 900 Rads in a single dose, which qualifies as reduced exposure (lethality < 100%). The lethal dose varies between strains of mice, and 900 Rads TBI is optimal for the adult C57BL/6 strain, as used in this protocolF.
    2. Following irradiation, recipient animals must be placed on an antibiotic/analgesic-supplemented drinking water regimen for 1 week; this must be outlined in your laboratory’s approved animal experimentation protocol.

Day 5: Quantification and transplantation of transduced bone marrow cells


  1. Isolation and lineage depletion of helper bone marrow cells
    1. Isolate bone marrow from 2 hind limbs of a wild-type adult mouse of the same strain/genetic background (congenic) as the animals used throughout the protocol. Follow Steps B1-B11 of the bone marrow isolation protocol outlined on Day 2.
      Notes:
      1. Isolation of bone marrow from 2 hind limbs (tibia + humeri only) of an adult mouse (approximately 8 weeks of age) after red blood cell lysis yields 50 x 106-70 x 106 cells.
      2. Many laboratories chose to bypass this step altogether, while others employ the complement-mediated T-cell depletion approach (when donor and recipient mice are 100% syngeneic). Due to the transfer of mice between laboratories and varyingly stringent approaches to backcrossing, we recommend the slightly more cumbersome approach of using lineage-depleted helper cells to avoid any possible immunological responses in the donor-recipient transplantation setting.
    2. Perform absolute cell quantification using the method of choice (manual cell quantification using a hemocytometer and Trypan blue exclusion or an automated cell counter; the former being the preferred method).
    3. Perform magnetic bead-based lineage depletion of helper bone marrow following the manufacturer’s instructions (https://www.miltenyibiotec.com/US-en/resources/technical-documents/data-sheets.html). This approach relies on negative selection, i.e., unlike positive CD117-cell enrichment (Day 2 Section B) collect the flow-through from the magnetic column into a 15 ml conical bottom tube placed on ice. The total expected yield of the Lin- fraction from 2 hind limbs of one adult mouse is 5 x 106 cells.
    4. Pellet the cells at 500 rcf at 4 °C for 5 min.
    5. Resuspend the Lin- cells in 1 ml SM and quantify the absolute live cell number by trypan blue exclusion using a hemocytometer (see Step A2). Keep cells on ice until ready to proceed with the bone marrow transplantation step below.
    6. Gently pulse-vortex the helper bone marrow to obtain a uniform single-cell suspension. Transfer the appropriate number of cells to a fresh tube 15 ml tube: you will need 2 x 105-3 x 105 helper bone marrow cells per recipient mouse (i.e., if 10 recipient mice are being used for the transplant, collect a volume containing the equivalent of 2 x 106-3 x 106 cells).
    7. Proceed to Step B13 of Day 2 of the protocol.

  2. Harvesting and quantification of transduced bone marrow stem and progenitor cells
    1. In a laminar flow cabinet, resuspend pMSCV-NOTCH1-L1601P-ΔP transduced bone marrow progenitor cells using a 1 ml pipettor (Day 3 Step B7). Pool cell suspensions from the 24-well plate in a 15 ml/50 ml conical tube.
    2. Pellet cells by centrifugation at 500 rcf at room temperature for 5 min.
    3. Resuspend cells in a known volume of OptiMEM transduction medium without growth factors and quantify the absolute number of live cells by manual counting using a hemocytometer and Trypan blue exclusion of dead cells. The resuspension volume is guided by the size of the cell pellet (approximately 5 ml if cells were initially harvested from all bones).
    4. Transfer two individual 100 µl aliquots of the cell suspension to 1.5 ml Eppendorf tubes for staining and quantification (this aliquot should contain no fewer than 50,000-100,000 cells to ensure accurate quantification in the next step). Keep the suspension of transduced cells in a 37 °C TC incubator until quantification is complete.
      Note: Loosen the screwcap of the 15 ml/50 ml conical collection tube to allow free air flow.
    5. When using the recommended PE-Cyanine7-conjugated anti-Sca-1 antibody, to a 100 µl aliquot of cells add 500 µl of SM and 1 µl of the anti-Sca-1 antibody. To the second aliquot of cells, add 500 µl of SM only (this negative control allows more accurate discrimination of the Sca-1-positive versus the Sca-1-negative population when performing subsequent flow cytometry analysis).
      Note: When using alternative fluorochrome conjugates, all antibodies should be titrated prior to use, although a 1:200 dilution works well for the majority of commercially available anti-Sca-1 conjugates. The 1:600 dilution in this step has been empirically established for the specific antibody listed in this protocol (see Materials and Reagents).
    6. Stain cells on ice for 45-60 min in the dark.
    7. Add 1 ml of SM to wash cells and centrifuge at 500 rcf for 5 min at 4 °C. Be very careful when aspirating the supernatant as the pellet may be quite small.
    8. Resuspend the pellet in 500 µl SM + 25 µl CountBright quantification beads. Add 5 µl of DAPI (pre-diluted to 0.5 mg/ml in H2O) for dead cell exclusion.
      Note: When using CountBright beads, use a pipettor to thoroughly resuspend the cells and make careful note of the final resuspension volume as this will be used in the next step to calculate the absolute number of transduced cells. Similarly, be sure to thoroughly vortex the CountBright beads and note the exact volume added for quantification and the number of beads/µl in the Lot # of CountBright beads being used.
    9. Run samples on a flow cytometer to determine the frequency of transduced GFP+ Sca-1+ cells. Be sure to gate on live cells only (DAPI-) and use the “no anti-Sca-1 antibody” control to set the gate. Draw an additional gate on the population of CountBright beads (Figures 2A-2D).
      Note: The live cell population (DAPI-) will contain a mix of GFP- (untransduced) cells and GFP+ (transduced) cells. The expected percentage of successfully transduced bone marrow stem and progenitor cells (Sca-1+) should be no less than 10-15% (GFP+ Sca-1+ double-positive cells). See Notes section for additional comments.


      Figure 2. Flow-cytometry based quantification of retrovirally transduced bone marrow progenitor cells. The figure depicts the stepwise gating strategy for A. Total in vitro expanded CD117+-enriched cells and CountBright quantification beads; B. doublet exclusion; C. dead cell exclusion (DAPI-); and D. frequency of transduced hematopoietic stem and progenitor cells (HSPCs: GFP+ Sca-1+).

    10. Calculate the absolute number of GFP+ Sca-1+ double-positive cells based on the number of cells in the target population and the number of CountBright beads collected. Follow the manufacturer’s instructions for cell quantification (see Data analysis and Figure 5).
    11. Remove cells from the TC incubator (Step B3 of Day 5) and resuspend by pipetting up and down/gently pulse-vortexing to obtain a uniform single cell-suspension.
    12. Remove an aliquot of transduced cells based on the calculations performed in Steps B4-B10 of Day 5. You will need 100,000 GFP+ Sca-1+ cells/recipient mouse. We have previously established that transplantation of 100,000 transduced cells results in 70-80% tumor penetrance (it is not recommended to exceed 150,000 GFP+ Sca-1+ cells as this results in tumor development from multiple clones). For example: if 10% of transduced cells are GFP+ Sca-1+ and the total cell suspension contains 1.5 x 106 cells/ml, you will need 0.67 ml/recipient animal. Therefore, if transplanting 10 recipient animals, 6.7-10 ml of total cell suspension is required for transplantation.
    13. Pool together the appropriate volume of transduced Sca-1+ GFP+ cells from the previous step and the required volume of helper bone marrow prepared in Step A6 of Day 5 above. The volume of helper bone marrow is dictated by the concentration of the cell suspension and the target number of recipient mice (use 2 x 105-3 x 105 helper bone marrow cells per recipient mouse).
    14. Pellet cells at 500 rcf at 4 °C for 5 min.
    15. Thoroughly resuspend the cells in sterile cold PBS (stored at 4 °C, or pre-chilled on ice) and pass through a 40 µm filter to remove debris/clumps. The total volume of PBS is dictated by the number of recipient animals, i.e., when performing transplantation by retro-orbital injection, resuspend cells in 100-50 µl PBS/recipient mouse.
    Note: It is recommended to use a 20% excess of cells resuspended in an equivalent 20% excess volume (i.e., if transplanting n = 10 recipient mice, prepare a cell suspension sufficient for n = 12 recipient mice). Remember to collect any remaining cell suspension from the bottom side of the 40 µm filter using a 1 ml pipettor as the entire volume of cell suspension will not pass through the strainer by gravity pull alone when using such as small volume.

  3. Transplantation of retrovirally transduced bone marrow cells
    The result of the final step above is a single-cell suspension containing a mixture of transduced GFP+ Sca-1+ cells (100,000/recipient) + un-transduced progenitor cells + Lin- helper bone marrow cells.
    1. Anesthetize irradiated recipient animals (Day 4; Section C) by intraperitoneal injection of 100 µl ketamine/xylazine cocktail using a 1 ml insulin syringe (see Recipes).
      Note: Alternatively, inhalational anesthesia with isoflurane is commonly used, though this has a very short recovery period and is not recommended for users without extensive prior experience.
    2. Once animals are fully sedated, inject 50-100 µl cell suspension into the retro-orbital sinus using a 0.5 ml 29 G½ insulin syringe (Yardeni et al., 2011).
      Note: Please refer to Yardeni et al. (2011) for a comprehensive technical overview of retro-orbital injections in mice.
    3. Closely monitor animals and maintain on antibiotic-supplemented drinking water for 7 days

Day 21: Detection of transient CD4+ CD8+ double-positive preleukemia cells in peripheral blood


Notes:

  1. Any method of blood collection must be approved by the local animal care committee and be outlined in your laboratory’s approved animal experimentation protocol. Only trained personnel may perform this procedure.
  2. This protocol recommends blood sampling by submandibular puncture, which is a rapid technique that minimizes trauma to experimental animals (Figure 3).

  1. If assessing CD4 and CD8 surface expression (optional), prepare 50 µl antibody cocktail/peripheral blood sample. When using the antibodies recommended in this protocol (see Materials and Reagents) dilute the antibodies in SM as follows: anti-CD4-APC 1:400 and anti-CD8-PeCy7 1:800. Keep the antibody cocktail in the dark at 4 °C until ready to proceed with staining.
  2. Prepare 1.5 ml Eppendorf tubes containing 15 µl Heparin (n = number of recipient mice on Day 5 above).
  3. Collect 2-4 drops/mouse of peripheral blood by submandibular puncture using a 5 mm lancet into the previously prepared collection tube containing heparin (Figure 3).


    Figure 3. Blood sampling by submandibular puncture. A. The “freckle” aids in correct localization of the submandibular vein. B. Collection of 2-4 drops of blood. C. Maximum recommended volume of peripheral blood obtained during this procedure.

  4. Prepare 4 ml round-bottom FACS tubes containing 1 ml ACK lysis buffer.
  5. Transfer approximately 75 µl of peripheral blood into ACK buffer and incubate at room temperature for 5 min.
  6. Add 2 ml SM and pellet the cells at 500 rcf at room temperature for 5 min.
  7. Resuspend the cell pellet in 1 ml ACK buffer and repeat the lysis and wash steps 3 times until red blood cells are thoroughly lysed (the final pellet may remain pink rather than white; however, it is not advisable to reduce the number of lysis steps as red blood cells are very “sticky” and often result in clogging of the flow cytometer).
  8. After the final wash step, resuspend the pellet in 50 µl antibody cocktail and incubate on ice for 45 min in the dark.
  9. Wash cells in 1 ml SM and pellet at 500 rcf for 5 min. Resuspend in 500 µl SM supplemented with DAPI (5 µg/ml final concentration).
  10. Analyse samples using a flow cytometer instrument fitted with a Blue 488 nm (GFP), Green 561 nm laser (PE-Cy7), Violet 405 nm (DAPI) and Red 633 nm (APC) laser.


    Figure 4. Monitoring development of NOTCH1-induced T-ALL. A. Percentage of GFP+ pre-leukemia blasts in peripheral blood. B. Preleukemia blast-cell CD4/CD8 surface expression 21 days after transplantation of NOTCH1-L1601P-ΔP retrovirally transduced murine bone marrow stem and progenitor cells. C. Comparative Kaplan-Meier survival curves in weeks following transplantation of 100,000 NOTCH1-L1601P-ΔP (solid black), 100,000 pMSCV empty vector controls (dashed black) and 50,000 ΔE-NOTCH1-pMSCV-transduced murine hematopoietic stem and progenitor cells (grey).

Data analysis

Quantification of the absolute number of GFP+ Sca-1+ double-positive cells, Day 5 (Figure 2 and Figure 5)
Quantification using CountBright Absolute Counting Beads for flow cytometry is performed according to the manufacturer’s instructions. The following values are required for this calculation:
https://www.thermofisher.com/order/catalog/product/C36950

  1. The absolute number of acquired GFP+ Sca-1+ cells (Figure 2D; Step B9 of Day 5).
  2. The absolute number of acquired beads (Figure 2A; Step B9 of Day 5).
  3. The absolute # of CountBright beads added to the sample (Step B8 of Day 5).
  4. The volume of SM in which cells were resuspended for flow cytometry analysis (Step B8 of Day 5).
  5. The volume of the aliquot of bone-marrow cell suspension used for quantification (Step B4 of Day 5).
  6. The absolute volume in which bone marrow cells were resuspended (Step B3 of Day 5).
  7. Calculate the absolute number of GFP+ Sca-1+ double-positive cells in the culture.


    Figure 5. GFP+-Sca1+ absolute quantification. CountBright bead-based approach to quantification of the absolute number of pMSCV-NOTCH1-L1601P-ΔP-GFP transduced hematopoietic stem and progenitor cells (see above and follow manufacturer’s instructions).

Notes

  1. In this protocol, replication-defective retrovirus encoding the human NOTCH1 oncogene and a traceable GFP marker (green fluorescent protein; this could alternatively be RFP, an antibiotic resistance gene, etc.) is produced by transient co-transfection of 293T cells with pCL-Eco (a plasmid that expresses the gag-pol-env packaging functions). The 293T cells produce an ecotropic retrovirus specific for mouse cells, but not entirely ineffective for human cells, necessitating stringent observation of BSL-2 biohazard conditions.
  2. This protocol was optimized using the jetPEI transfection reagent. Alternative transfection systems may be used successfully but will require individual optimization.
  3. Here we outline the generation of oncogenic NOTCH1-L1601P-ΔP-driven tumors. Due to the > 15kb size of this retroviral construct, it is difficult to achieve transduction efficiency of bone marrow progenitor cells that exceeds 10% (GFP+ Sca-1+). A robust 70-80% frequency of transformation is achieved upon transplantation of 100,000 transduced progenitor cells, which is preceded by 45 ± 20% GFP+ pre-leukemia blasts in peripheral blood on day 21 (Figures 4A and 4B). Tumor progression commonly results in mortality 12-25 weeks post-transplant, reflecting kinetically more variable tumor development when compared to ΔE-NOTCH1-driven T-ALL (Figure 4C). When using the highly oncogenic constitutively active ΔE-NOTCH1 allele, transduction efficiency of HSCs falls in the 30 ± 10% range, resulting in 100% transformation efficiency. Subsequent T-ALL development occurs 6-8 weeks after transplantation of 50,000 GFP+ Sca-1+ progenitor cells, with GFP+ pre-leukemia blasts in the peripheral blood 21 days post-transplant reaching 60-70% and consequently 0% survival exceeding 8 weeks (Figure 4C). This protocol has also been successfully employed to generate alternative mouse models of leukemia, such as ΔE-NOTCH1, c-Myc or Flt3-ITD overexpression-induced T-ALL and AML leukemia, with 100% penetrance upon transduction of Lin- C57BL/6-derived bone marrow cells and subsequent transplantation of 50, 000 transduced cells with 100% penetrance by 12 weeks (previously published data), illustrating it’s efficacy across multiple lineage-specific oncogenes.

Recipes

  1. Staining Medium (SM)
    PBS
    2% FBS
    1 mM Hepes
    0.5 µM EDTA
  2. 293T growth/transfection medium
    DMEM
    10% FBS
    100 U/ml penicillin G
    100 µg/ml streptomycin
  3. Growth-factor supplemented CD117+ cell culture/transduction medium
    Opti-MEM I Reduced Serum Medium, GlutaMAX Supplement
    10% FBS
    100 U/ml penicillin
    100 µg/ml streptomycin
    55 µM 2-mercaptoethanol (β-mercaptoethanol)
    10 ng/ml recombinant mouse IL-3
    10 ng/ml recombinant mouse IL-6
    25 ng/ml recombinant mouse IL-7
    50 ng/ml recombinant mouse Flt3-L
    50 ng/ml recombinant mouse SCF
    25 ng/ml recombinant mouse TPO
  4. Ketamine/xylazine anesthesia cocktail (1 ml)
    150 µl Ketamine (100 mg/ml)
    150 µl Xylazine (20 mg/ml)
    700 µl PBS

Acknowledgments

This work was supported by the National Institute of Health grants R35 CA210065 (AF) and R01 CA155743 (AF). AAW was supported by a Rally Foundation fellowship. We thank D. Herranz for critical review of the protocol.

Competing interests

The authors declare no conflict of interest.

Ethics

All animals used in the development of this protocol were housed in specific pathogen-free facilities at the Irving Cancer Research Center at the Columbia University Medical Center, New York, NY, in accordance with NIH guidelines for the Care and Use of Laboratory Animals. All animal procedures were approved by the Columbia University Institutional Animal Care and Use Committee (IACUC).

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简介

[摘要 ] T细胞急性淋巴细胞白血病(T-ALL)是一种侵袭性血液恶性肿瘤,其起源于T细胞引发的造血祖细胞的转化。尽管T-ALL是一种异质且分子复杂的疾病,但超过65%的T-ALL患者在NOTCH1 基因中带有激活突变。大多数与T-ALL相关的NOTCH1 突变要么破坏负调控区,允许在没有配体结合的情况下激活信号,要么导致蛋白酶体降解终止NOTCH1信号终止所涉及的C末端PEST域被截短。迄今为止,逆转录病毒转导模型在很大程度上依赖于侵袭性截短的变种的过度表达。 NOTCH1 (例如ICN1或ΔE-NOTCH1)可导致信号传导的超生理水平,并且在人类T-ALL中很少见。当前方案描述了小鼠骨髓分离,造血干细胞和祖细胞(HSC)富集,然后逆转录病毒转导的致癌突变体形式的NOTCH1受体(NOTCH1-L1601P-ΔP)的方法,该方法与获功能突变最常见于患者样品中。组成型活性NOTCH1的这种强制表达的标志是胸腺外未成熟T细胞发育的瞬时波,此波在致癌性转化为T-ALL之前。此外,该方法通过允许白血病细胞与微环境之间的串扰来模拟体内白血病的转化和进展,这是基于细胞系的体外研究无法解释的一个方面。因此,HSC转导和移植模型更真实地概括了人类疾病的发展,为进一步的体内和离体功能研究提供了高度全面和通用的工具。

[背景 ] T细胞急性淋巴细胞白血病(T-ALL)是一种侵袭性血液肿瘤,是由造血祖细胞向T细胞发育引发的恶性转化而引起的。T-ALL占儿童ALL(急性淋巴细胞白血病)的15%和成人ALL(急性淋巴细胞白血病)的25%,并且男性发病率高于女性(Goldberg 等,2003 )。尽管以高度异质性和分子复杂性为特征(Liu 等人,2017 ),但仍有超过65%的T-ALL患者在NOTCH1 基因中携带功能获得性突变(Weng 等人,2004 )。因此,NOTCH1信号转导的组成型激活是T细胞转化中最主要的致癌途径。

NOTCH1蛋白去velopmentally保守的I型跨膜受体是胸腺细胞发育过程中发挥的T细胞谱系的承诺和细胞生长和增殖的显着指导作用(瑞克等人,1999年; Defto 和贝文,2000 )。NOTCH1信号传导是在相邻细胞表面表达的跨膜配体结合后开始的。这种相互作用促使ADAM10金属蛋白酶切割受体的胞外域,然后在受体的跨膜域中切割γ-分泌酶。从胞内结构域(ICN1)的核和靶基因的转录活化的易位膜结果随后的释放(高班和Ilagan,2009;安德森等人,2011 )。最后,NOTCH1信号的终止受C端PEST域的磷酸化调控,该磷酸化靶向ICN1进行泛素化和FBXW7介导的蛋白酶体降解(O'Neil 等,2007; Thompson 等,2007 )。人T-ALL中NOTCH1的组成性激活是由于严格调控该多步过程的机制破坏所致。大多数T-ALL相关突变导致负调控区的破坏,在没有配体结合的情况下导致NOTCH1信号激活,或者C端PEST域被截短,从而使ICN1逃避蛋白酶体降解,因此破坏信号传导的终止(Weng 等,2004 )。在过去的十年中,强效联合化疗方法的引入使小儿T-ALL的治愈率提高到近90%。尽管如此,原发性难治性或复发性T-ALL患者的预后仍然很差(Goldberg 等人,2003;Litzow 和Ferrando ,2015 ),强调需要进一步解读T-ALL转化的分子病理学,鉴定更多特定的治疗目标,以及开发更有效,毒性更小的多药疗法。

迄今为止,体外人类T-ALL衍生的细胞系培养仍然是研究T-ALL的最常用实验方法。值得注意的是,该模型无法校正与十年培养期相关的遗传漂移,也无法解释白血病细胞与肿瘤微环境之间的串扰(Passaro 等人,2015; Pitt 等人,2015 )。因此,动物模型为功能研究提供了另一种方法,可以更准确地在体内概括人类T-ALL 。小鼠造血干细胞(HSC)逆转录病毒转导和移植模型已广泛应用于模仿NOTCH1驱动的T-ALL的启动和进展(Wendorff 等,2010; Medyouf 等,2011; Gachet 等,2013 )。研究表明,ICN1或ΔE-NOTCH1(受体的胞外域截短形式)在鼠骨髓祖细胞中的逆转录病毒表达可驱动胸腺非依赖性T细胞发育并迅速诱导T-ALL发育(Pear 等, 1996; Perez-Garcia 等人,2013 )。然而,应该指出的是,在人T-ALL中很少发现NOTCH1的这些积极地被截短的变体,导致信号传导活性的超生理水平(Chiang 等,2008 )。实际上,ΔE-NOTCH1截短的等位基因仅在携带染色体易位的一小部分患者中表达(1%)(Ellisen 等,1991;Palomero 等,2006 )。更常见的是,人类T-ALL具有获得的功能获得点突变,这种突变主要发生在细胞外HD和C末端PEST域内(Weng 等,2004 )。值得注意的是,有20%的T-ALL患者同时出现这两种突变驱动的激活机制,它们产生的活性水平远低于ΔE-NOTCH1 (Chiang 等,2008 )。由于NOTCH信号转导的结果在许多发育环境中高度依赖剂量(Artavanis-Tsakonas 等人,1999; Bray,2016 ),我们假设活性水平对白血病的发生过程具有相似的影响。当前协议描述了一种用致癌突变体NOTCH1受体(NOTCH1-L1601P-ΔP)进行小鼠造血干细胞(HSC)逆转录病毒转导的方法,该方法与患者样品中发现的功能获得性突变极为相似。L1601P-ΔP突变体包含一个常见的HD点突变(L1601P),该突变可实现不依赖配体的激活(Malecki 等,2006 ),以及一个移码突变,导致氨基酸2,473-2,555被截断,其中包括负调控因子PEST域(ΔP)。我们概述了从小鼠中分离骨髓细胞,富集造血干细胞和祖细胞,然后进行逆转录病毒转导并移植到同基因受体中的方法(图1)。在该模型中,组成型活性突变体NOTCH1的强制表达通常会导致移植后3周出现胸腺T细胞淋巴细胞生成波,这表现为GFP + 白血病前CD4 + CD8 + 双阳性(DP)未成熟T细胞的短暂出现在外周血中(图4)。导致致命性白血病进展的转化发生在移植后12-15周内(Wendorff et al。,2019 )(图4)。当与组成型或诱导型敲除转基因模型组合时,可以轻松修改协议以解决候选基因在NOTCH1驱动的T-ALL转化,肿瘤起始和肿瘤进展中的作用(Wendorff et al。,2019 )。此外,该方法为潜在的新型抗白血病药物的临床前评估提供了快速有效的模型系统(Herranz 等,2015; Sanchez-Martin 等,2017 )。

关键字:小鼠模型, 体内, T细胞急性淋巴细胞白血病, NOTCH1 信号, NOTCH1 突变, 白血病形成, 逆转录酶病毒, 转化, 造血干细胞, 骨髓, 移植

材料和试剂


 


移液器技巧
封口膜
1层Kimwipes 精致任务刮水器(Kimberly-Clark Professional,货号:06-666)
150毫米TC处理过的细胞培养皿(Corning,Falcon,目录号:353025)
24孔TC处理的微孔板(Corning,Falcon,货号:353847)
5 ml圆底聚苯乙烯管(Corning,Falcon,目录号:352008)
15 ml锥形底部离心管(Corning,Falcon,目录号:352196)
50 ml锥形底部离心管(Corning,Falcon,目录号:352070)
3 ml BD Luer-Lok 吸头注射器(Becton Dickinson,目录号:309657)
30毫升BD Luer-Lok 尖头注射器(Becton Dickinson,目录号:302832)
25毫米无菌PES注射器过滤器,孔径为0.45 µm(Thermo Fischer Scientific,目录号:7252545)
40 µm细胞过滤器(Corning,Falcon,目录号:352340)
70 µm细胞过滤器(Thermo Fisher Scientific,目录号:22-363-548)
Amicon Ultra-15离心15 ml样品量过滤器NMWL 100KDa(EMD Millipore ,目录号:UFC910008)
U-100胰岛素注射器,1毫升,26克(BD Biosciences,目录号:329652)
U-100胰岛素注射器,0.5毫升,29克½(Exelint 国际,目录号:26028)
金毛动物柳叶刀,5毫米(Medipoint ,目录号:NC9891620)
用于阴性细胞选择的LS色谱柱(Miltenyi Biotec ,目录号:130-042-401)
6-12周龄的C57BL / 6骨髓供体小鼠和同龄C57BL / 6骨髓受体小鼠(至少6周龄)(杰克逊实验室,目录号:000664)
293T细胞(ATCC,目录号:CRL-32 16)
pCL- Eco逆转录病毒包装载体(Addgene ,目录号:12371)(Naviaux 等,1996 )
pMSCV-NOTCH1-L1601P-ΔP (Chiang 等,2008 )
JETPEI DNA转染试剂提供用150mM 的NaCl 溶液(Polyplus ,目录号:101-10N)
PBS:不含钙和镁的细胞培养磷酸盐缓冲盐水(1x)(Corning,目录号:21040CV)
DMEM(Corning,目录号:10-013-CV)
Opti-MEM I降低血清培养基,GlutaMAX 补充剂(Gibco ,目录号:51985)
胎牛血清(FBS),热灭活和无菌(Sigma-Aldrich,目录号:F4135)
青霉素(10,000 IU)和链霉素(10,000 µg / ml),工作浓度为100倍(Corning,目录号:30002CI)
HEPES 1 M(Gibco,货号:15630080)
0.5 M EDTA(Lonza,货号51201)
β- 巯基乙醇,55 mM (Gibco ,目录号:21985023)
台盼蓝(Gibco,货号:15250061)
ACK裂解缓冲液(Quality Biologicals,目录号:118-156-721)
CD117小鼠微珠(Miltenyi Biotec ,目录号:130-091-224)
小鼠谱系细胞耗减试剂盒(Miltenyi Biotec ,目录号:130-090-858)
聚乙烯感染/转染试剂(EMD密理博,目录号:TR-1003-G)
用于流式细胞术的CountBright 绝对计数珠(Invitrogen,目录号:C36950)
重组鼠IL-3(Peprotech ,目录号213-13)
重组鼠IL-6(Peprotech ,目录号:216-16)
重组鼠IL-7(Peprotech ,目录号217-17)
重组鼠Flt3-L(Peprotech ,目录号:250-31L)
重组鼠SCF(Peprotech ,目录号:250-03)
重组鼠TPO(Peprotech ,目录号315-14)
抗小鼠Sca-1(Ly-6A / E)单克隆抗体(克隆D7),PE-Cyanine7共轭物(eBioscience ,目录号:25-5981)
(可选)抗小鼠CD4单克隆抗体(克隆RM4-5),APC偶联物(BD Biosciences,目录号:561091)
(可选)抗小鼠CD8单克隆抗体(克隆53-6.7),PE-Cyanine7共轭物(BD Biosciences,目录号:561097)
DAPI(4',6-Diamidino-2-Phenylindole,Dilactate)(Invitrogen,目录号:D3571)
肝素,1,000 USP单位/ ml(Sagent Pharmaceuticals,目录号:25021-400-30)
盐酸氯胺酮注射液(100 mg / ml:受控物质); 咨询机构的兽医
赛拉嗪无菌溶液(20 mg / ml:AKORN Animal Health;目录号:139-236); 咨询机构的兽医
染色介质(SM;请参见食谱)
293T培养/转染培养基(请参阅食谱)
补充生长因子的转导培养基(请参见食谱)
氯胺酮/甲苯噻嗪麻醉鸡尾酒(请参阅食谱)
 


设备


 


1 ml / 200 µl / 20 µl 移液器
研钵和研杵
细胞培养培养箱,37 °C / 5%CO 2
解剖工具:剪刀,镊子,手术刀刀片(推荐来源:Fine Science Tools www.finescience.com)
血细胞计数器(推荐)或自动细胞计数器
层流生物安全柜
配有离心桶转子和平板适配器的冷冻离心机
MACS MultiStand (Miltenyi Biotec ,目录号:130-042-303)
MidiMACS 分离器(Miltenyi Biotec ,目录号:130-042-302)
带有绿色荧光蛋白(GFP)激发/发射滤光片的荧光显微镜
具有蓝色(488 nm),紫色(405 nm),绿色(561 nm)和红色(633 nm)激光的流式细胞仪仪器(推荐:LSRFortessa ,BD Biosciences)
伽马射线源或替代性小动物辐照器
 


软件


 


FlowJo(FlowJo LLC,美国俄勒冈州阿什兰)
GraphPad Prism软件(美国加利福尼亚拉荷亚,www.graphpad.com)
 


程序


 


逆转录病毒转导和骨髓移植的这种方法可用于过表达任何目的致癌基因,从而在小鼠中产生淋巴样或髓样白血病。转化效率,疾病进展动力学和肿瘤穿透性将取决于所用致癌基因的致瘤性和实验变量,例如移植的转导的造血干细胞的数量。


注意:请参阅说明部分该协议的额外的信息,并请参阅图4 为所述的比较的pMSCV-NOTCH1-L1601P-ΔP 和ΔE-NOTCH1 白血病模型(推荐的细胞数,白血病发展和外显率的动力学)。


 


这里描述的程序已经过优化,可用于建模由NOTCH1受体的L1601P-ΔP突变体形式特别驱动的T-ALL发育。例如,当使用较大的构建体,例如pMSCV-NOTCH1-L1601P-ΔP-ires-GFP(> 15 kb)时,对293T细胞的转染进行了修饰,以解决降低的转染效率,并抵消了病毒滴度的降低。此外,该协议还建议除胫骨和股骨外,还应从髋骨和脊椎中分离出骨髓。这种方法大大增加了可用于后续转导的骨髓干细胞和祖细胞的总数,同时减少了所需实验动物的数量。尽管此协议中列出的所有小鼠(供体和受体小鼠)均处于C57BL / 6背景上,但如果骨髓供体和受体小鼠处于同基因遗传背景下,则可以使用其他小鼠品系。但是,重要的是要记住,每个菌株都有独特的背景等位基因,它们可能与突变,转基因或其他遗传插入物相互作用并改变其表达(https://www.jax.org/news-and-insights/2006/ 6月/基于小鼠的生物医学研究中的遗传背景的重要性)。


 


请注意,在此协议中概述的实验方法需要动手活性(日6连续天小号0 - 5)中,用随后的随访第21天(图小号1A 和1个B;图4) 。


 


重要提示:所有实验程序都必须经过当地动物保健委员会的批准。


笔记:


在开始实验方案之前,请根据所需的受体动物数量计算所需的骨髓供体小鼠数量。作为“经验法则”,仅从时收获骨髓2个后腿,n = 1个的骨髓供体是足够用于移植成n = 2 - 3 接受者动物; 从所有骨骼(后肢和前肢,骨盆骨和脊柱)收获骨髓时,n = 1的骨髓供体足以移植到n = 6-8的接受动物中。
在方案的第5天,还需要另一只未经治疗的同基因野生型小鼠作为辅助性骨髓细胞的来源。
 


D:\ Reformatting \ 2020-3-2 \ 1902520--1179 Agnieszka Wendorff 729498 \ Figs jpg \图1.jpg


图1.从小鼠造血干细胞生成致癌的NOTCH1驱动的T-ALL的实验设计。一。关键实验步骤的卡通表示。乙。实验策略的时间表。


 


293T细胞的瞬时转染


第0天:


在转化前24-48 h接种293T细胞,其密度应达到转染时约60-70%的融合度(对于293T培养基;请参见食谱)。解冻新鲜小瓶的活冻n细胞后,使用传代不少于2次的293T 细胞。总共不得超过20个段落。仅使用经过测试的支原体阴性细胞(Young 等,2010 )。


注意:应根据培养板的大小和用于生产病毒的293T细胞的生长动力学来优化接种密度。以下值已针对150 mm组织培养板进行了优化,其中293T培养板与供体小鼠和用于骨髓分离的总骨数的保守比例为1:1。


 


第一天:


在晚上,进行293T细胞的瞬时转染(表1);有关一般指南,请参见https://www.polyplus-transfection.com/wp-content/uploads/2015/09/CPT_101_jetPEI_vU.pdf。


在无菌条件下,在单独的试管中制备DNA和转染混合物:
 


 


 


表1. 每1x 150 mm 293T细胞板推荐的转染试剂和逆转录病毒构建体的量(根据需要扩大)


管#1


每150毫米板的数量


jetPEI 转染试剂


50微升


氯化钠


450微升


总容积/板


500微升


 


 


2号管


每150毫米板的数量


pMSCV-NOTCH1-L1601P-ΔP逆转录病毒构建体


20微克


pCL- 生态包装结构


15微克


NAC 升


X微升


填充至总体积/板


500微升


 


将1号试管的内容物添加至2号试管(始终向DNA混合物中添加转染试剂,反之亦然),轻轻涡旋并在室温下静置20-30分钟。
将混合物逐滴添加至293T细胞;每150毫米平板1毫升。
孵育过夜(12-16小时)。
 


第二天:


293T细胞的瞬时转移,继续
在早晨,除去转染培养基,并轻轻用13-15毫升新鲜293T培养基更换。放在培养箱中直到第二天。


笔记:


一个。293T细胞将在转染后12小时内开始产生GFP。为了确认成功的转染并获得转染效率的定性评估,建议在转染后16-24小时使用荧光显微镜验证GFP的产生;转染的产病毒细胞呈现出明亮的荧光。         


b。进行所有培养基更换时要格外小心,因为细胞已被转染试剂敏化,并通过连续培养更容易从平板上脱离。        


C。从此以后,在处理BSL-2危险材料时,请遵守所有安全要求。       


 


分离骨髓细胞和CD117 + 细胞富集
笔记:


该方案建议通过使用研钵和研杵压碎骨头来隔离骨髓,这种方法可产生更高和更一致的细胞产量,同时保持高细胞活力。对于新手研究人员来说,从髋骨和脊椎中分离骨髓显得比较麻烦,但它极大地提高了每只小鼠的细胞产量,并减少了提取骨髓所需的实验动物的数量,这是使用转基因小鼠模型时的关键考虑因素。尽管从实验小鼠中解剖胫骨和股骨在该领域已经很成熟,但获得髋骨和小腿椎骨的情况却很少见。我们强烈建议您在采用这种方法之前,先阅读由Lo Celso和Scadden (2007)发布的视频教程。
小鼠骨髓细胞的逆转录病毒转导最常使用总谱系阴性骨髓细胞的阴性选择进行。如此处所述,对CD117 + 细胞的阳性选择使造血干细胞的转导规模增加,这导致具有自我更新潜能的目标人群的转化效率提高。
始终将染色培养基(SM)和骨髓细胞悬液(BM)放在冰上。在4 °C下执行所有离心步骤。
以下提供的值基于从n = 1只成年小鼠(约8-10周龄)的所有骨骼中分离出的总BM,并应根据骨髓供体动物的目标数量适当放大。
 


牺牲供体小鼠:建议进行CO 2 窒息,然后颈脱位。
解剖每只供体小鼠的胫骨,股骨和骨盆骨;总的预期骨髓细胞产量约为120 x 10 6 /只动物,但随供体动物的年龄而变化。如果需要,可以通过额外隔离脊椎将总细胞产量提高到160 x 10 6 -200 x 10 6 /动物。
使用手术刀刮擦肌肉组织,清洁骨骼。使用Kimwipes /纸巾擦拭手指之间的骨头,以去除残留的组织。建议通过脱位而不是切割骨头来轻柔地折断膝盖骨和其他关节的软骨,因为这样可以保留骨头的完整性,提高产量并降低污染的风险。如果要隔离脊椎,请将椎骨分成3-4个单独的部分,然后除去脊髓。用Kimwipes 擦拭彻底清洁。将清洁的骨头放在冰上的冷SM中。
尽管不是绝对必要,但建议在无菌条件下在层流生物安全柜中执行所有后续步骤,以防止污染。使用研钵和研杵在5毫升冷SM中将骨头粉碎(粉碎而不是研磨,以保持高细胞活力)。反复抽吸并使用3 ml注射器弹出细胞悬液,以生成单细胞悬液,然后将其通过40 µm细胞过滤器进入置于冰上的50 ml收集管中。向研钵中加入5 ml SM,并重复此过程约3次,直到骨头被完全压碎并呈现半透明/白色。
在最后一轮压碎后,用5 ml SM洗涤滤网;这导致每只供体小鼠约40 ml单细胞骨髓悬浮液。
在4 °C下以500 rcf的速率沉淀细胞7分钟。
吸出/倒出SM(沉淀物将显示为红色)。
通过将沉淀物重悬于5 ml ACK缓冲液中进行红细胞裂解,并在室温下孵育5分钟。使细胞悬液通过70 µm细胞过滤器进入置于装有至少20 ml SM的冰上的50 ml锥形管中,以终止裂解反应,然后用SM充满该管,以进一步稀释ACK裂解缓冲液。
在4 °C下以400 rcf的速率沉淀细胞10分钟。
吸出/倒出上清液(沉淀现在应该出现在WHITE附近),并小心地将细胞沉淀重悬于5 ml冷SM中。加入15 ml冷SM并轻轻脉冲涡旋以获得20 ml均匀的细胞悬液。
通过使用血球计数器或自动细胞计数器进行手动细胞计数,确定存活细胞的数量(锥虫蓝排除)。
注意:由于总骨髓细胞的异质性,前一种方法可以更准确地量化细胞数量。


按照制造商的说明(https://www.miltenyibiotec.com/US-en/resources/technical-documents/data-sheets.html ),继续使用抗CD117微珠进行CD117 + 阳性细胞选择。在最后一步中,洗脱到15 ml Eppendorf管中,然后加入10 ml SM。沉淀细胞,在4 ℃下在500 5分钟RCF 。
将CD117 + 细胞富集的沉淀重悬于2 ml冷SM中,并进行细胞定量。将细胞放在冰上。
在该方案的此步骤中,建议使用血细胞计数器手动计数细胞并用锥虫蓝排除死细胞,以确保准确的细胞计数。当从所有骨骼中收获细胞时(如上所述),总预期产量约为5 x 10 6 CD117 + 细胞/供体动物。
在密度为0.5 x 10 6 细胞/ ml的2 ml / 孔补充生长因子的培养基(对于GF培养基,请参见食谱)中,在24孔组织培养板上富集CD117 + -的细胞,并培养过夜。
 


第3天:第一轮病毒收集和骨髓祖细胞转导


 


首次收集和浓缩293T培养物中的病毒上清液
注意:在2级生物安全柜中于室温下执行以下步骤,并遵守处理BSL-2有害物质的所有安全要求。


收集并过滤病毒上清液:
从293T培养物中收集病毒上清液(VSN),并通过70 µm过滤器进入50 ml锥形收集管中以除去大细胞碎片。
在第4天第二次收集VSN,用13-15 ml新鲜293T培养基仔细补充培养物。
通过将VSN穿过0.45 µm PES注射器过滤器来清除小碎片和聚集物(最容易的方法是将VSN从50 ml锥形收集管中抽吸到装有60 G ,18 G½ 规格大针头的30-60 ml注射器中,向上旋转注射器以防止溢出和污染,并在将VSN喷射到新的50 ml圆锥形底部收集管中之前用注射器过滤器更换针头)。
注意:两个连续的过滤步骤将除去所有可能导致下一步堵塞Amicon Ultra浓缩柱的颗粒。


病毒上清液浓度
将VSN转移到Amicon 过滤柱管中,以达到最大容量(Amicon Ultra = 15 ml)。
在室温下以2500 rcf的速度离心,直到约80%的VSN通过了过滤单元(典型的最终浓缩物体积/ 15mL VSN体积= 200µl)。如果从> 1 x 150毫米293T产病毒皿中收集了VSN,请使用相同的过滤单元重复步骤A2a-A2b ,直到达到所需的VSN最终体积。将最终体积的浓缩VSN转移到Eppendorf管中。丢弃Amicon 过滤装置。
注意:为含有2ml的CD117 + 富集的骨髓的24孔培养板的每个孔产生100 µl VSN(浓度为20x)。我。例如,如果在方案第2天将富含CD117 +的骨髓细胞接种在12孔中,则将VSN浓缩至最终体积为1200 µl。用新鲜的OptiMEM 细胞培养基根据需要调节体积。切记添加适量的聚烯烃(下面的步骤B1 )。


 


首次转导富含CD117 +的骨髓祖细胞
注意:将离心机预热以进行旋转感染至28 °C 。将减速速度(中断)设置为0。


向装有浓缩VSN的Eppendorf管中加入Polybrene试剂,使其终浓度为80 µg / ml(20x)。
将100 µl含20x Polybrene的20x VSN加到含有2ml GF培养基的CD117 + 富集的骨髓细胞的24孔板的每个孔中。
用石蜡膜密封板,以防止旋转感染过程中离心机受到污染。
                                                        在28 °C下以1,100 rcf 离心90分钟。
小心取出封口膜,然后将板放回TC培养箱中。让细胞恢复至少6小时。
6-8小时后,轻轻取出1/2的介质, 约 1毫升(轻轻倾斜平板;使用1毫升移液器,并在吸液时保持移液器吸头与培养基表面齐平,以防止吸出骨髓细胞)。
加入1 ml新鲜的GF补充转导培养基,并使细胞恢复过夜(12-16小时)。
 


第4天:第二轮病毒收集和骨髓祖细胞转导


 


第二次从293T培养物中收集和浓缩病毒上清液
在协议的第3天,重复步骤A1a-A1c和A2a-A2b 。


 


富含CD117 +的骨髓祖细胞的第二次转导,先前在第3天进行转导,并在添加GF的培养基中培养过夜。
在实验方案的第3天重复步骤B1-B7 。


 


辐照受体小鼠
笔记:


尽管可以接受6-12周的范围,但建议使用8周龄的小鼠作为受体。由于应在移植前至少8小时对受体小鼠进行辐照,因此该方案建议在移植前一天的晚上进行辐照。单次剂量可提供900 Rads的全身剂量(TBI),相当于减少暴露(致死率<100%)。致死剂量因小鼠品系而异,如本规程中所用,900 Rads TBI最适合成年C57BL / 6品系。
辐照后,必须将接受动物的动物置于抗生素/止痛剂补充的饮用水中1周;必须在您实验室批准的动物实验规程中对此进行概述。
 


第5天:转导的骨髓细胞的定量和移植


 


辅助骨髓细胞的分离和谱系耗竭
从与整个实验方案中使用的动物相同的品系/遗传背景(同基因)的野生型成年小鼠的两个后肢中分离出骨髓。遵循第2天概述的骨髓分离方案的步骤B1-B11 。
笔记:


从2个后肢骨髓的隔离(胫骨+ 呜呜ERI 只)后红细胞裂解的产率50成年小鼠(约8周龄)的×10 6 - 7个0 ×10 6 细胞。
许多实验室选择完全绕开此步骤,而另一些实验室则采用补体介导的T细胞耗竭方法(供体和受体小鼠是100%同基因的)。由于实验室之间的小鼠转移和回交的严格方法不同,我们建议使用谱系耗竭的辅助细胞稍微麻烦一些的方法,以避免在供体-受体移植环境中发生任何可能的免疫反应。
使用所选方法进行绝对细胞定量(使用血细胞计数器和台盼蓝排除法或自动细胞计数器进行手动细胞定量;前者是首选方法)。
按照制造商的说明(https://www.miltenyibiotec.com/US-en/resources/technical-documents/data-sheets.html )执行基于磁性珠的辅助骨髓沿袭耗竭。该方法依赖于阴性选择,即与阳性CD117细胞富集(第2天B 部分)不同,它将从磁柱收集的流通液收集到置于冰上的15 ml锥形底管中。的总预期产量的大号中- 从一个成年小鼠的2个后肢分数为5×10 6 细胞。
将细胞在500 rcf 和4 °C下沉淀5分钟。
重悬林- 在1ml SM细胞,并通过使用血球台盼蓝排除量化的绝对活细胞数目(参见步骤A2 )。将细胞保持在冰上,直到准备进行下面的骨髓移植步骤为止。
轻轻地将辅助骨髓脉动涡旋以获得均匀的单细胞悬液。将适当数量的细胞转移到一个15 ml的新鲜试管中,每只受体小鼠需要2 x 10 5 -3 x 10 5个辅助骨髓细胞(即,如果将10只受体小鼠用于移植,则收集一个包含相当于2 x 10 6 -3 x 10 6个单元的体积)。
前进到步骤B13 的第2天的协议。
 


收集和定量转导的骨髓干细胞和祖细胞
在层流柜中,使用1 ml移液器重悬pMSCV-NOTCH1-L1601P-ΔP转导的骨髓祖细胞(第3天,步骤B7 )。在15 ml / 50 ml锥形管中合并来自24孔板的细胞悬液。
通过在室温下500 rcf 离心5分钟来沉淀细胞。
在没有生长因子的已知体积的OptiMEM 转导培养基中重悬细胞,并通过使用血球计数器和台盼蓝排除死细胞进行手动计数来定量活细胞的绝对数量。重悬液的体积取决于细胞沉淀的大小(如果最初从所有骨骼中收获细胞,则约为5 ml)。
将细胞悬液的两个100μl等分试样转移到1.5 ml Eppendorf管中进行染色和定量(该等分试样应包含不少于50,000-100,000个细胞,以确保在下一步中进行准确定量)。将转导细胞的悬浮液保存在37 °C TC培养箱中,直到完成定量。
注意:松开15 ml / 50 ml锥形收集管的螺帽,使空气自由流通。


当使用推荐的PE-Cyanine7偶联的抗Sca-1抗体时,向100微升细胞等分试样中加入500微升SM和1微升抗Sca-1抗体。在第二等分试样的细胞中,仅添加500 µl SM(此阴性对照可在进行后续流式细胞术分析时更准确地区分Sca-1阳性人群和Sca-1阴性人群)。
注意:使用替代的荧光染料偶联物时,所有抗体应在使用前进行滴定,尽管1:200的稀释度对于大多数市售抗Sca-1偶联物效果很好。对于此协议中列出的特定抗体,已根据经验确定了此步骤中的1:600稀释度(请参见材料和试剂)。


在黑暗中在冰上将细胞染色45-60分钟。
加入1 ml SM洗涤细胞,并在4 °C下以500 rcf 离心5分钟。吸出上清液时要非常小心,因为沉淀可能很小。
将沉淀重悬于500 µl SM + 25 µl CountBright 定量珠中。加入5 µl DAPI(在H 2 O中预先稀释至0.5 mg / ml )以排除死细胞。
注意:使用CountBright 珠子时,请使用移液器彻底重悬细胞,并仔细记录最终的重悬体积,因为该体积将在下一步中用于计算转导细胞的绝对数量。同样,请确保将CountBright 珠子充分涡旋,并注意所添加的准确体积用于定量,以及所使用的CountBright 珠子批号中的珠子数量/微升。


在流式细胞仪上运行样品,以确定转导的GFP + Sca-1 + 细胞的频率。一定要在活细胞门只(DAPI - ),并使用“无抗的Sca-1抗体”控制旋钮设置门。在CountBright 珠的总体上绘制一个额外的门(图s 2A- 2 D)。
注:活细胞群(DAPI - )将包含GFP的混合- (未转)细胞和GFP + (转)细胞。成功转导的骨髓干细胞和祖细胞(Sca-1 + )的预期百分比应不少于10-15%(GFP + Sca-1 + 双阳性细胞)。请参阅注释部分以获取其他注释。


 


D:\ Reformatting \ 2020-3-2 \ 1902520--1179 Agnieszka Wendorff 729498 \ Figs jpg \图2.jpg


图2。基于流式细胞仪的逆转录病毒转导的骨髓祖细胞的定量。该图描述了A 的逐步门控策略。总的体外扩增CD117 + 富集细胞和CountBright 定量珠。B.双重排除;C.死细胞排除(DAPI - ); D.被转导的造血干细胞和祖细胞的频率(HSPC:GFP + Sca-1 + )。


 


根据目标群体中的细胞数和收集的CountBright 珠子数,计算GFP + Sca-1 + 双阳性细胞的绝对数。按照制造商的说明进行细胞定量(请参阅数据分析和图5 )。
除去单元F ROM中的TC培养箱(步骤B3 的第5天通过吹吸),并再悬浮和向下/轻轻脉冲- 涡旋得到均匀的单细胞悬浮液。
根据第5天的步骤B4-B10 中执行的计算,取出等分的转导细胞。您将需要100,000个GFP + Sca-1 + 细胞/收件人小鼠。我们先前已经确定,移植100,000个转导的细胞会导致70-80%的肿瘤渗透率(不建议超过150,000个GFP + Sca-1 + 细胞,因为这会导致多个克隆形成肿瘤)。例如:如果10%的转导细胞是GFP + Sca-1 + ,并且总细胞悬浮液包含1.5 x 10 6个细胞/ ml,则您将需要0.67 ml /收件人动物。因此,如果要移植10只受体动物,则需要6.7-10 ml的总细胞悬浮液进行移植。
池一起适当体积transduc ED的Sca-1 + GFP + 来自前一步骤中制备的辅助骨髓所需体积的细胞的步骤A6 的第5天AB OVE。辅助骨髓的体积由细胞悬液的浓度和受体小鼠的目标数量决定(每只受体小鼠2 x 10 5 -3 x 10 5个辅助骨髓细胞)。
在4 °C下以500 rcf沉淀细胞5分钟。
将细胞彻底重悬在无菌的冷PBS中(储存于4 °C 或在冰上预冷),然后通过40 µm过滤器除去碎屑/团块。PBS的总体积由受体动物的数量决定,即,通过眼眶后注射进行移植时,将细胞重悬于100-50 µl PBS /受体小鼠中。
注意:建议使用20%过量的细胞重悬于等效20%的体积中(即,如果移植n = 10只受体小鼠,则准备足以容纳n = 12只受体小鼠的细胞悬液)。切记使用1 ml移液器从40 µm过滤器的底部收集所有残留的细胞悬液,因为使用小体积时,仅靠重力拉动,整个细胞悬液就不会通过滤网。


 


逆转录病毒转导的骨髓细胞的移植
上述的最后一个步骤的结果是含有GFP转导的混合物的单细胞悬浮液+ 的Sca-1 + 细胞(100,000 /接受者)+未转导的祖细胞+林- 辅助骨髓细胞。


通过使用1 ml胰岛素注射器腹膜内注射100 µl氯胺酮/ 甲苯噻嗪鸡尾酒来麻醉受辐照的动物(第4天;C 节)。  
注意:另外,尽管异氟醚的恢复期非常短,但不建议具有广泛先验经验的用户使用,但通常使用异氟烷进行吸入麻醉。


给动物完全镇静后,使用0.5 ml 29 G½ 胰岛素注射器将50-100 µl细胞悬液注入眼眶后窦(Yardeni 等,2011 )。
注意:请参阅Yardeni 等。(2011)对小鼠进行眼眶后注射的全面技术概述。


严密监视动物并维持补充抗生素的饮用水7天。
 


第21天:检测外周血中的瞬时CD4 + CD8 + 双阳性白血病前细胞


 


笔记:


任何采血方法都必须经过当地动物保健委员会的批准,并在实验室批准的动物实验方案中进行概述。只有经过培训的人员才能执行此过程。
该方案建议通过下颌下穿刺采血,这是一种快速的技术,可以最大程度地减少对实验动物的伤害(图3 )。
 


如果评估CD4和CD8表面表达(可选),请准备50 µl抗体混合物/外周血样品。当使用此方案中推荐的抗体时(请参见材料和试剂),按如下方法稀释SM中的抗体:抗CD4-APC 1:400和抗CD8-PeCy7 1:800。将抗体混合物在4 °C 的黑暗环境中放置,直到准备进行染色。
准备含有15 µl肝素的1.5 ml Eppendorf管(n = 上文第5天的受体小鼠数量)。
使用5 mm刺血针通过下颌下穿刺收集2-4滴/小鼠外周血,进入先前准备好的装有肝素的收集管中(图3)。
 


D:\ Reformatting \ 2020-3-2 \ 1902520--1179 Agnieszka Wendorff 729498 \ Figs jpg \图3.jpg


图3.下颌下穿刺采血。一。“雀斑”有助于下颌下静脉的正确定位。乙。收集2-4滴血。Ç 。在此过程中获得的最大推荐外周血量。


 


准备包含1 ml ACK裂解缓冲液的4 ml圆底FACS管。
将约75 µl的外周血转移到ACK缓冲液中,并在室温下孵育5分钟。
加入2 ml SM并在室温下以500 rcf 沉淀细胞5分钟。
将细胞沉淀重悬于1 ml ACK缓冲液中,并重复裂解和洗涤步骤3次,直到红细胞完全裂解为止(最终沉淀可能保持粉红色而不是白色;但是,建议不要减少裂解步骤,因为红细胞非常“粘”,经常导致流式细胞仪堵塞。
最后的洗涤步骤后,将沉淀重悬于50 µl抗体混合物中,并在冰上于黑暗中孵育45分钟。
用1 ml SM洗涤细胞,并以500 rcf 沉淀5分钟。重悬于500μlSM,再加DAPI(终浓度5μg/ ml)。
使用配有蓝色488 nm(GFP),绿色561 nm激光(PE-Cy7),紫色405 nm(DAPI)和红色633 nm(APC)激光的流式细胞仪仪器分析样品。
 


D:\ Reformatting \ 2020-3-2 \ 1902520--1179 Agnieszka Wendorff 729498 \ Figs jpg \图4.jpg


图4.监视NOTCH1诱导的T-ALL的发展。一。外周血中GFP + 白血病前胚细胞的百分比。乙。NOTCH1-L1601P-ΔP逆转录病毒转导的鼠骨髓干细胞和祖细胞移植后21天,白血病前胚细胞CD4 / CD8表面表达。Ç 。移植100,000个NOTCH1 -L1601P-ΔP(纯黑色),100,000 pMSCV 空载体对照(黑色虚线)和50,000ΔE-NOTCH1-pMSCV所转导的小鼠造血干细胞和祖细胞(灰色)移植后几周的比较Kaplan-Meier生存曲线。


 


数据分析


 


第5天,对GFP + Sca-1 + 双阳性细胞的绝对数量进行定量(图2 和图5)


根据生产商的说明,使用CountBright 绝对计数珠进行流式细胞仪定量。此计算需要以下值:


https://www.thermofisher.com/order/catalog/product/C36950


获取的GFP的绝对数量+ 的Sca-1 + 细胞(图2D; 步骤B9 的第5天)。
的获取的珠的绝对数量(图2A; 步骤B9 的第5天)。
的绝对#CountBright 珠添加至样品(步骤B8 的第5天)。
其中细胞再悬浮,用于流式细胞术分析SM的体积(步骤B8 的第5天)。
骨髓细胞悬浮液的等分试样用于定量体积(步骤B4 的第5天)。
绝对量,其中骨髓细胞再悬浮(步骤B3 的第5天)。
计算培养物中GFP + Sca-1 + 双阳性细胞的绝对数量。
 


D:\ Reformatting \ 2020-3-2 \ 1902520--1179 Agnieszka Wendorff 729498 \ Figs jpg \图5.jpg


图5. GFP + -Sca1 + 绝对定量。基于CountBright 珠的方法可定量pMSCV-NOTCH1-L1601P-ΔP-GFP转导的造血干细胞和祖细胞的绝对数量(请参见上文,并遵循制造商的说明)。


 


笔记


 


1. 在此协议中,通过将293T细胞与T细胞进行瞬时共转染,产生了编码人NOTCH1 癌基因和可追踪GFP标记(绿色荧光蛋白;也可以是RFP,抗生素抗性基因等)的复制缺陷型逆转录病毒。pCL -Eco(表达gag-pol-env包装功能的质粒)。Ť 他293T细胞产生的亲嗜性反转录病毒特异性的小鼠细胞,但不是完全无效的人类细胞,这需要的BSL-2的生物危害的条件严格观察。      


2. 使用jetPEI 转染试剂优化了该方案。替代转染系统可能会成功使用,但需要进行个别优化。      


3. 在这里,我们概述了致癌的NOTCH1-L1601P-ΔP驱动的肿瘤的产生。由于该逆转录病毒构建体的大小> 15kb,因此很难实现超过10%(GFP + Sca-1 + )的骨髓祖细胞的转导效率。鲁棒7 变换的0-80%频率100000个导的祖细胞,其通过45±20%GFP之前的移植后达到+ 预白血病胚细胞在外周血中在第21天(图小号4A 和4 B)。肿瘤进展通常会导致移植后12至25周的死亡率,与ΔE-NOTCH1驱动的T-ALL相比,反映出动力学上更具可变性的肿瘤发展(图4C)。当使用高度致癌的组成型活性ΔE-NOTCH1等位基因时,HSC的转导效率在30±10%范围内,导致100%的转化效率。随后的T-ALL发育发生在移植50,000个GFP + Sca-1 + 祖细胞后6-8周,而GFP + 白血病在移植后21天的白血病前胚细胞达到60-70%,因此存活率超过0% 8周(图4C)。该方案也已成功用于生成白血病的其他小鼠模型,例如ΔE-NOTCH1,c - Myc 或Flt3-ITD过表达诱导的T-ALL和AML白血病,转导Lin - C57BL / 6 时具有100%的渗透性来源的骨髓细胞,然后在12周内移植50,000个具有100%渗透率的转导细胞(先前发表的数据),说明了其在多种谱系特异性癌基因中的功效。      


 


菜谱


 


染色介质(SM)
PBS


2%FBS


1 mM 麻疹


0.5 µM EDTA


293T生长/转染培养基
记忆体


10%FBS


100 U / ml青霉素G


100 µg / ml链霉素


补充生长因子的CD117 + 细胞培养/转导培养基
Opti-MEM I降低血清培养基,GlutaMAX 补充剂


                                          10%FBS


100 U / ml青霉素


100 µg / ml链霉素


55 µM 2-巯基乙醇(β- 巯基乙醇)


10 ng / ml重组小鼠IL-3


10 ng / ml重组小鼠IL-6


25 ng / ml重组小鼠IL-7


50 ng / ml重组小鼠Flt3-L


50 ng / ml重组小鼠SCF


25 ng / ml重组小鼠TPO


氯胺酮/甲苯噻嗪麻醉混合物(1毫升)
150 µl氯胺酮(100 mg / ml)


150微升赛拉嗪(20毫克/毫升)


700微升PBS


 


致谢


 


由美国国立卫生研究院支持这项工作授予R35 CA210065(AF)和R01 CA155743(AF)。AAW得到了Rally基金会奖学金的支持。我们感谢D. Herranz 对协议的严格审查。


                                                                                   


利益争夺


 


作者宣称没有利益冲突。


 


伦理


 


根据NIH《实验动物的护理和使用指南》,将用于制定该方案的所有动物都安置在纽约州哥伦比亚大学医学中心欧文癌症研究中心的特定无病原体设施中。所有动物程序均获得哥伦比亚大学机构动物护理和使用委员会(IACUC)的批准。


 


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引用:Wendorff, A. A. and Ferrando, A. A. (2020). Modeling NOTCH1 driven T-cell Acute Lymphoblastic Leukemia in Mice. Bio-protocol 10(10): e3620. DOI: 10.21769/BioProtoc.3620.
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