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Construction of NSG-CTL Mice
用NSG小鼠建立NSG-CTL模型   

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Abstract

The NSG-CTL mouse model is a humanized mouse model that allows the generation of peripheral human immune responses, particularly CD8+ Cytotoxic T lymphocyte (CTL) responses, and serves as an effective model for studying gene-based therapies. Natural antigen-specific T cell responses in humanized mice are relatively weak and this model was developed to boost antigen specific responses, in this case to HIV, to more closely assess these responses in vivo. We have engineered human T cells that develop in these mice to express a molecularly cloned T cell receptor (TCR) specific to HIV. Cloned TCRs to any antigen can theoretically be used to study specific responses in vivo, as long as the tissue that is manipulated is of the same human leukocyte antigen (HLA) type. The modification of hematopoietic stem cells with antigen specific TCRs allows the development of mature, functional T cells in the periphery of these mice that are specific to that antigen following normal developmental processes. This model has recently been published (Kitchen et al., 2012) and this was a major modification of the Humanized Mouse BLT model published by Melkus et al. (2006). We use Non-obese diabetic (NOD)-Severe Combined Immunodeficient (SCID), common Gamma chain knockout (γc-/-)—or NSG—mice, and implant fetal thymus pieces along with genetically modified CD34+ hematopoietic stem cells (HSCs), isolated from fetal liver, under the kidney capsule to develop into a functional thymic implant. At the same time, we deplete the mouse’s bone marrow by total body irradiation and inject more modified HSCs intravenously for hematopoietic engraftment in the mouse bone marrow. This protocol outlines the procedures to process the fetal tissue, genetically transduce the HSCs using lentiviral vectors expressing a molecularly cloned T cell receptor, and perform the subcapsular kidney implant surgery.

Processing Fetal Thymus and Isolating CD34+ Cells from Fetal Liver

Materials and Reagents

  1. Fetal thymus/liver pair from 18 – 22 week gestation specimen
  2. Dulbecco's phosphate buffered saline (PBS) (Life Technologies, catalog number: 14190 )
  3. 1x Iscove's Modified Dulbecco's Medium (IMDM) (Life Technologies, catalog number: 12440 )
  4. RPMI Medium 1640 – L-Glutamine (Life Technologies, catalog number: 11875 )
  5. Number 11 scalpels
  6. Collagenase type IV, (Life Technologies, InvitrogenTM, catalog number: 17104-019 ) 50,000 U/ml in PBS diluted 1:10 in ddH2O
  7. Hyaluronidase (240,000 U/ml in 1:10 PBS) (Sigma-Aldrich, catalog number: H6254 )
  8. DNase I (30,000 U/ml in 1:10 PBS) (Worthington, catalog number: 2006)
  9. Pip/Tazo (Zosyn) – 45 mg/ml is 100x stock
  10. Ficoll-Paque PLUS (GE Healthcare, catalog number: 17-1440 )
  11. Fetal bovine serum (FBS) (Omega Scientific, catalog number: FB-12 )
  12. CD34 MicroBead Kit, human (Miltenyi Biotec, catalog number: 130-046-702/3 )

Equipment

  1. Class II biosafety cabinet
  2. 37 °C incubator
  3. Shaker
  4. Centrifuge
  5. 100 mm Petri dishes
  6. 100 μm cell strainer
  7. 10 ml syringes
  8. 16 gauge blunt end needle
  9. 0.22 μm filter
  10. 50 ml conical tubes
  11. 15 ml conical tubes
  12. T25 and T150 flask

Procedure

  1. An overview of the procedure is noted in Figure 1.
    Note: all steps to be performed in a Class II biosafety cabinet or in a sterile, HEPA-filtered environment.


    Figure 1.

  2. Wash the thymus in a 15 ml conical tube: Pour off the medium into a beaker of bleach, using a scalpel to prevent the thymus from sliding into the bleach. Fill the tube to 15 ml with PBS. Cap and invert several times to wash the thymus. Decant supernatant into the bleach. Repeat 3-4 times.
  3. Add 7 ml RPMI + 10% FBS and decant everything into a 100 mm Petri dish.
  4. With two scalpels, cut the thymus into small (~1 mm2) pieces. If you need to phenotype/ genotype your tissue, such as for HLA type*, remove some media containing free-floating thymocytes at this point for analysis. *HLA typing can be performed at low resolution by flow cytometry [we typically identify HLA-A*02+ Tissue), and genotyping for high resolution identification (i.e. HLA-A*0201) is performed by a clinical immunogenetics laboratory or by a PCR based analysis kit (Life Technologies, InvitrogenTM, catalog number: 470214D)].

  5. Pipette media and thymus pieces into a T25 flask. Supplement media with 450 μg/ml Pip/Tazo and gently rock the flask back and forth to mix. Culture overnight at 37 °C. This step is important in preventing bacterial contamination of tissue to be implanted into mice.
  6. Wash the liver in a 50 ml conical tube: Pour off the medium into a beaker of bleach, using a scalpel to prevent the liver from sliding into the bleach. Fill the tube with PBS. Cap and invert several times to wash the liver. Decant supernatant into bleach. Repeat 3-4 times.
  7. Add 10 ml IMDM and decant everything into a 100 mm Petri dish.
  8. With two scalpels, cut the liver into small (~3 mm2) pieces. If you find white connective tissue, scrape the red liver tissue from it and discard it in the bleach.
  9. Homogenize the tissue: suck the liver pieces and media into a 10 ml syringe fitted with a 16 gauge blunt needle, and transfer to a 50 ml conical tube. Suck up into syringe and expel one more time to fully homogenize tissue.
  10. Prepare enzymes and Pip/Tazo: Add 200 μl each of the Collagenase, Hyaluronidase, DNase, and Pip/Tazo to 10 ml IMDM in a new tube.
  11. Draw enzyme/media mixture into a 10 ml syringe, fit it with a 0.22 μm filter, and filter it directly into the cell suspension.
  12. Cap the suspension tube and seal with Parafilm on the outside to prevent leaks. Rotate or gently shake by machine at 37 °C for 90 min.
  13. Pipet the digested cell suspension through a 100 μm cell strainer into a fresh 50 ml tube.
  14. Add PBS to the suspension to bring the volume up to 50 ml. Split this into two tubes of 25 ml.
  15. Underlay the cells with 10 ml Ficoll-Paque per tube by dispensing the Ficoll very slowly with a 10 ml pipet at the bottom of the cell suspension. Spin at 950 x g for 20 min at 21 °C without brake.
  16. The interface should be thick. Suck it out with a 5 ml pipet and transfer to another 50 ml tube. Then, equally split interface between 250 ml tubes.
  17. You should have two tubes of interface. Bring the volume of each up to 50 ml with PBS. Spin at 240 x g at 21 °C for 10 min. Aspirate supernatant carefully.
  18. Combine the two pellets as you wash three more times with 50 ml PBS containing 2% FBS.
  19. Resuspend the pellet in 50 ml RPMI + 10% FBS. Count your cells.
  20. Optional: Culture cells in a T150 flask or two at a concentration of 10 X 106 c/ml for 1 – 2 h to let adherent cells adhere. Supplement media with 450 μg/ml Pip/Tazo. Count cells after this incubation.
  21. Sort CD34+ cells using Miltenyi Biotec's CD34 MicroBead Kit, human, according to the manufacturer's protocol. Count cells. Culture the CD34+ cells at a concentration between 1-10 x 106 c/ml in RPMI + 10% FBS at room temperature until you are ready to transduce them.
  22. Culture the CD34- fraction overnight at 37 °C at a concentration of 10 x 106 c/ml in RPMI + 10% FBS supplemented with 450 μg/ml Pip/Tazo.

Transducing CD34+ Cells

Materials and Reagents

  1. RetroNectin (Clontech, catalog number: T100A/B ) 20 μg/ml in PBS (aliquot and freeze 1 mg/ml stock at -20 °C)
  2. Dulbecco's phosphate buffered saline (PBS) (Life Technologies, catalog number: 14190 )
  3. FACS Buffer (4% FBS in PBS)
  4. Infection medium – 2% Human Serum Albumin (American Red Cross, catalog number: NDC 52769-451-05) in Yseel's Serum-Free T-Cell Medium (Gemini, catalog number: 400-102 ) (Optional: Supplement with 50 ng/ml human MGDF, SCF, and Flt3-ligand)
  5. Cytokine medium – RPMI with 10% FBS, supplemented with 100 ng/ml human IL-3, IL-6, SCF
  6. Lentiviral vector, titered
  7. Cell Scraper

Equipment

  1. Biosafety cabinet
  2. 37 °C incubator
  3. Centrifuge 
  4. 6-well Plate, Non-Treated (BD Biosciences, Falcon®, catalog number: 351146 )

Procedure

  1. Calculate the number of transduction wells required of a 6 well-tissue culture plate (1 well can handle no more than 8 X 106 cells). Coat the needed number of wells of a non-treated 6-well plate with RetroNectin: Dispense 1.25 ml of RetroNectin solution (20 μg/ml in PBS) into each well and allow the covered plate to stand for 2 h at room temperature in a clean biosafety cabinet.
  2. Remove RetroNectin solution from wells.
  3. Add 1.25 ml of FACS buffer to each well for blocking. Allow the plate to stand at room temperature for 30 min.
  4. Remove FACS Buffer and wash wells once with PBS.
  5. Keep PBS in the coated wells until the plate is ready for use. You can store the plate at 4 °C. Remove PBS from the wells immediately before plating cells.
  6. For transduction efficiency controls, aliquot ~1 x 105 untransduced CD34+ cells and culture in 200 μl Cytokine Medium in 96-well plate for 5-7 days at 37 °C.
  7. Plate remaining CD34+ cells in Infection Medium in RetroNectin-coated wells (~2 x 106 cells/ml) and incubate at 37 °C for one hour (see Alternate method in Step 9 below).
  8. Add your vector to the wells at a multiplicity of infection (MOI) between 2–10. Gently mix and incubate overnight at 37 °C.
  9. Alternate method (for lower-efficiency vectors): Add infection medium and vector to wells first, and spin at 3,000 x g for 90-120 min at 12 °C, then add cells in medium on top of this, gently swirl to mix, and incubate overnight at 37 °C.
  10. The following morning, harvest the cells: Gently scrape the bottoms of the wells, collect cells, and count.
  11. Aliquot ~1 x 105 cells from each condition and culture in 200 μl cytokine medium in 96-well plate for 5-7 days at 37 °C. After 5-7 days, check transduction efficiency by flow cytometry, looking at your reporter, for instance, using the previously cultured, untransduced aliquot (see step 6) as your control.
  12. For each mouse you will transplant, you will need 0.5 x 106 transduced CD34+ cells per implant, and 0.5 x 106 transduced CD34+ cells for injection.
  13. For the implanted cells, combine 0.5 x 106 transduced CD34+ cells per mouse with 4.5 x 106 CD34- cells per mouse, aliquot into sterile 1.5 ml screw-cap tubes. Spin the cells to pellet them, aspirate supernatant, spin them again and aspirate any remaining supernatant, and keep the dry pellets on ice.
  14. For the injected cells, spin 0.5 x 106 transduced CD34+ cells per mouse to pellet them, aspirate supernatant, and resuspend them in plain RPMI, 150 μl per mouse. Keep these on ice.

Tissue Transplants to Construct NSG-CTL Mice

Materials and Reagents

  1. Fetal thymus pieces
  2. CD34+ cells for implantation and injection
  3. NSG Mice, at least 6–8 weeks old
  4. Matrigel, High Concentration (BD Biosciences, catalog number: 354248 )
  5. Ketamine (Ketaject (Phoenix Pharmaceuticals, catalog number: NDC 57319-542-02), 100 mg/ml) / Xylazine (AnaSed Injection (Lloyd Laboratories, catalog number: NADA 139-236), 100 mg/ml), 2.5% each in PBS
  6. Isoflurane
  7. Betadine
  8. Sterile saline
  9. Carprofen (Rimadyl (Pfizer, catalog number: NADA 141-199)) diluted 1:100 in sterile saline (keep dilution at 4 °C for up to 24 h)
  10. PBS

Equipment

  1. Flow Cytometer
  2. 15 ml conical tubes
  3. Sterile gauze pads
  4. 60 mm dish
  5. 15 ml conical tube
  6. Positive Displacement Pipette (1-20 μl) and Tips
  7. Oster clipper with No. 40 blade
  8. 60 mm Petri dish 
  9. Isopropanol wipes
  10. 16 gauge cancer implant needle with a round-filed tip (cannula and trocar)
  11. Surgical tools: Needle-tipped forceps, curved forceps, scissors, hemostat, autoclip applier and wound clips
  12. Vicryl sutures, 4-0 (Owens & Minor, catalog number: 23000J304H)
  13. Insulin syringes
  14. 1 ml syringe

Procedure

  1. Up to 24 h prior to the surgery, perform total body irradiation on the mice to be used with a dose of 2.7 Gy.
  2. Pour thymus pieces and medium from the flask into a 60 mm dish. Pour some PBS into another dish.
  3. Fill a 1 ml syringe (no needle) with PBS.
  4. Chill positive displacement pipette tips by putting them in open sterile tubes in ice. Keep on ice with the dried cell pellets and Matrigel. It is important to keep the Matrigel and any tubes or tips that will touch it cold at all times until the implant needle is loaded.
  5. Anesthetize mice: Weigh mice and inject them interperitoneally with 15 μl of Ketamine/Xylazine per gram of body weight.
  6. When the mice have become sluggish from the Ketamine/Xylazine, place an animal's nose in a 15 ml conical tube that is stuffed with gauze soaked in isoflurane until the animal loses consciousness, its breathing slows to about half the normal rate, and it no longer responds to a paw pinch. Keep this tube on hand during the surgery to administer more isoflurane if the breathing starts to become more rapid.
  7. Punch the animal's ears for numbering or use your own preferred identification method.
  8. Using the Oster clipper with No. 40 blade, shave the left side of each mouse from hip to shoulder between the center of the back and the belly.
  9. Subcutaneously inject 0.3 ml of the diluted Carprofen up by the animal's shoulder, then place the mouse on a sterile dressing on its right side, facing left.
  10. Flush the cannula of the 16 gauge implant needle with PBS.
  11. Using a pair of needle-tipped forceps, place a piece of thymus from the dish into the opening of the cannula with the trocar just inside the opening, then pull back on the trocar to aspirate the tissue into the cannula.
  12. Use a positive displacement pipette and a chilled tip to put 5 μl of cold Matrigel into one tube with a dried cell pellet and gently stir just enough to get the cells into suspension. Do not pipette up and down. Pipette the Matrigel/cell suspension into the opening of the cannula as you slowly pull back on the trocar to load the needle. This is much easier with a helper to pipette while you manipulate the implant needle.
  13. Swab the bare skin of the mouse with Betadine, then wipe down the area with an isopropanol wipe two times.
  14. Determine the darkest spot under the skin, which indicates the location of the spleen. The kidney is approximately 5 mm dorsal to the spleen.
  15. Lift up the skin with curved forceps and make an incision about 15 mm long in the skin over the kidney parallel to the spleen. Make a similar cut in the peritoneum layer below.
  16. In male mice, the kidney should be visible, and can be extruded simply by pressing on the abdomen. You can support the kidney with a hemostat.
  17. In female mice, the ovary blocks the kidney. Using a hemostat, pick up the ovary and drag out the kidney.
  18. Use the needle-tipped forceps to pluck a tiny hole (1-2 mm) at the posterior end of the kidney capsule.
  19. Slide the implant needle into this hole and along the kidney until the opening of the cannula is completely covered by the kidney capsule.
  20. Gently extrude the cells and tissue under the kidney capsule, and pull the needle back out. The thymus pieces can be sticky, so you can use your curved forceps to make sure the thymus piece does not come out with the needle.
  21. Lift up the peritoneum with the forceps and gently use the hemostat to push the kidney back into place.
  22. Tie one stitch in the peritoneum with a double knot.
  23. Use two autoclips to close the skin.
  24. Mix the transduced CD34+ cells saved for injection and suck 150 μl (0.5 x 106 cells) up into an insulin syringe. Inject these cells into the mouse through retroorbital vein injection.
  25. Place one drop of PBS onto each eye and lay the mouse on its side back in a cage.
  26. When all the mice have been implanted, confirm that the animals are regaining consciousness before leaving them.
  27. Post-operational care: The day after the surgery, subcutaneously inject 0.3 ml of diluted Carprofen and 1.2 ml of sterile saline separately into each mouse. On days 2 and 3 following surgery, subcutaneously inject 1.5 ml of sterile saline into each mouse. Monitor the mice and the incisions for 10 days following surgery. Remove the autoclips and weigh the mice after 10 days.
  28. After 8-10 weeks, check engraftment by bleeding the mice and performing FACS analysis on the peripheral blood, staining for markers such as CD45, CD3, CD4, CD8, and any genes your vector should express. A typical flow cytometry profile, staining for human CD3 (T cell marker) and a transgenic, HIV specific TCR, is provided in Figure 2.


    Figure 2. Sample of TCR + Cells in te Peripheral Blood

  1. For a visual reference on a similar type of transplantation procedure, please see Vatakis et al., 2012 in the Journal of Visualized Experiments
    (http://www.jove.com/video/4181/using-blt-humanized-mouse-as-stem-cell-based-gene-therapy-tumor).

Variation

  1. Transduced CD34+ cells can be viably frozen instead of injected post-surgery. In this case, do not irradiate the mice prior to surgery. Instead, wait 4-6 weeks, thaw the cells, irradiate the mice (2.70 Gy), and inject the cells retro-orbitally, 0.5 x 106 cells/mouse. Post-operational care for these mice will be 0.3 ml Carprofen on the first day after surgery only. No saline injections will be required.

Acknowledgments

This work was funded by grants from the NIH (number R01AI078806 (S.G.K), the California HIV/AIDS Research Program (CHRP) (number 163893) (S.G.K.), the UC Multicampus Research Program and Initiatives from the California Center for Antiviral Drug Discovery (number MRPI-143226), and from funding by the UCLA Center for AIDS Research (CFAR) (number P30 AI28697).

References

  1. Kitchen, S. G., B. R. Levin, G. Bristol, V. Rezek, S. Kim, C. Aguilera-Sandoval, A. Balamurugan, O. O. Yang and J. A. Zack (2012). In vivo suppression of HIV by antigen specific T cells derived from engineered hematopoietic stem cells. PLoS Pathog 8(4): e1002649.
  2. Melkus, M. W., J. D. Estes, A. Padgett-Thomas, J. Gatlin, P. W. Denton, F. A. Othieno, A. K. Wege, A. T. Haase and J. V. Garcia (2006). Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1. Nat Med 12(11): 1316-1322. 
  3. Vatakis, D. N., Bristol, G. C., Kim, S. G., Levin, B., Liu, W., Radu, C. G., Kitchen, S. G. and Zack, J. A. (2012). Using the BLT humanized mouse as a stem cell based gene therapy tumor model. J Vis Exp (70): e4181.


简介

NSG-CTL小鼠模型是允许产生外周人免疫应答,特别是CD8 +细胞毒性T淋巴细胞(CTL)应答的人源化小鼠模型,并且作为研究基于基因的治疗的有效模型。人源化小鼠中天然抗原特异性T细胞应答相对较弱,并且开发这种模型以加强抗原特异性应答,在这种情况下是HIV,以更密切地评估这些体内的反应。我们已经设计在这些小鼠中发展的人T细胞,以表达对HIV特异性的分子克隆的T细胞受体(TCR)。克隆的TCR与任何抗原理论上可用于研究体内的特异性应答,只要所操作的组织是相同的人白细胞抗原(HLA)类型。具有抗原特异性TCR的造血干细胞的修饰允许在这些小鼠的外周中形成成熟的,功能性T细胞,其在正常发育过程之后对该抗原是特异性的。该模型最近已经发表(Kitchen等人,2012),这是Melkus等人发表的人源化小鼠BLT模型的主要修饰。 (2006)。我们使用非肥胖糖尿病(NOD) - 严重联合免疫缺陷(SCID),常见的γ链敲除(γc -/- )或NSG小鼠,并植入胎儿胸腺片与基因修饰的CD34 +造血干细胞从胎儿肝脏分离,在肾囊下发育成功能性胸腺植入物。同时,我们通过全身照射消耗小鼠的骨髓,并静脉内注射更多的修饰的HSC用于在小鼠骨髓中造血植入。该协议概述了处理胎儿组织的过程,使用表达分子克隆的T细胞受体的慢病毒载体基因转导HSC,并进行囊下肾移植手术。

处理胎儿胸腺和隔离CD34 +细胞从胎儿肝

材料和试剂

  1. 胎儿胸腺/肝脏对从18 - 22周妊娠标本
  2. Dulbecco's磷酸盐缓冲盐水(PBS)(Life Technologies,目录号:14190)
  3. 1x Iscove's Modified Dulbecco's Medium(IMDM)(Life Technologies,目录号:12440)
  4. RPMI Medium 1640-L-谷氨酰胺(Life Technologies,目录号:11875)
  5. 第11号手术刀
  6. 胶原酶IV型(Life Technologies,Invitrogen ,目录号:17104-019)在PBS中以1:10稀释于ddH 2 O中的50,000 U/ml
  7. 透明质酸酶(240,000U/ml,1:10 PBS)(Sigma-Aldrich,目录号:H6254)
  8. DNase I(30,000U/ml,1:10 PBS)(Worthington,目录号:2006)
  9. Pip/Tazo(Zosyn)-45mg/ml是100×储液
  10. Ficoll-Paque PLUS(GE Healthcare,目录号:17-1440)
  11. 胎牛血清(FBS)(Omega Scientific,目录号:FB-12)
  12. CD34 MicroBead试剂盒,人(Miltenyi Biotec,目录号:130-046-702/3)

设备

  1. II类生物安全柜
  2. 37℃孵育器
  3. 振动器
  4. 离心机
  5. 100mm培养皿
  6. 100μm细胞过滤器
  7. 10ml注射器
  8. 16号钝端针
  9. 0.22μm过滤器
  10. 50ml锥形管
  11. 15ml锥形管
  12. T25和T150烧瓶

程序

  1. 过程的概述如图1所示。
    注意:在II级生物安全柜或无菌,HEPA过滤的环境中执行的所有步骤。


    图1.

  2. 在15ml锥形管中洗涤胸腺:将培养基倒入烧杯中的漂白剂,使用解剖刀防止胸腺滑入漂白剂。用PBS将管填充至15ml。盖和倒转几次洗胸腺。将上清液倒入漂白液中。重复3-4次。
  3. 加入7ml RPMI + 10%FBS,并将所有东西倒入100mm培养皿中
  4. 用两个解剖刀将胸腺切成小(〜1mm)的片。如果你需要表型/基因型你的组织,如HLA类型*,删除一些媒体包含自由浮动的胸腺细胞在这一点进行分析。 * HLA分型可以通过流式细胞术以低分辨率进行(我们通常鉴定HLA-A * 02 +组织),并且用于高分辨率鉴定的基因分型(即 HLA-A * 0201)临床免疫遗传学实验室或通过基于PCR的分析试剂盒(Life Technologies,Invitrogen ,目录号:470214D)]。

  5. 将培养基和胸腺片吸入T25烧瓶中。补充介质与450微克/毫升的Pip/Tazo和轻轻摇动烧瓶来回混合。在37℃下培养过夜。该步骤对于防止植入小鼠的组织的细菌污染是重要的
  6. 在50 ml锥形管中洗涤肝脏:将培养基倒入烧杯中的漂白剂,使用手术刀防止肝脏滑入漂白剂。 用PBS填充管。 盖和倒转几次洗肝。 将上清液倒入漂白液中。 重复3-4次。
  7. 加入10 ml IMDM,并将所有东西倒入100 mm培养皿中。
  8. 使用两个解剖刀,将肝脏切成小块(〜3mm)。 如果发现白色结缔组织,从其中刮掉红色肝脏组织,并将其丢弃在漂白剂中
  9. 均质化组织:将肝片和培养基吸入装有16号钝针的10ml注射器,并转移到50ml锥形管中。 吸入注射器并再次排出以完全均匀组织
  10. 准备酶和Pip/Tazo:在新管中加入200μl每种胶原酶,透明质酸酶,DNase和Pip/Tazo到10ml IMDM。
  11. 将酶/培养基混合物倒入10ml注射器中,用0.22μm过滤器装配,并直接过滤到细胞悬浮液中。
  12. 盖住悬挂管,在外面用封口膜密封以防止泄漏。用机器在37℃下旋转或轻轻摇动90分钟。
  13. 将消化的细胞悬液通过一个100μm细胞过滤器移入新鲜的50ml管中
  14. 向悬浮液中加入PBS使体积达到50 ml。分成两个25毫升的管。
  15. 通过用在细胞悬液底部的10ml移液管非常缓慢地分配Ficoll,用每管10ml的Ficoll-Paque底下细胞。在无刹车的情况下,于21℃以950旋转20分钟。
  16. 界面应该厚。用5ml移液管取出,转移到另一个50ml管中。然后,在250ml管之间均等分开界面
  17. 你应该有两个接口管。用PBS将每种体积加至50 ml。在240℃下旋转10分钟,在240×g下旋转。小心吸出上清液。
  18. 当您用含有2%FBS的50ml PBS洗涤三次时,合并两个沉淀
  19. 将沉淀重悬在50ml RPMI + 10%FBS中。 计数您的单元格。
  20. 任选:在T150烧瓶中培养细胞或以10×10 6 c/ml的浓度培养细胞1〜2小时,以使粘附的细胞粘附。 补充培养基用450μg/ml Pip/Tazo。 计数细胞孵育后。
  21. 根据制造商的方案,使用Miltenyi Biotec的CD34 MicroBead试剂盒(人)分选CD34 +细胞。 计数单元格。 在室温下在RPMI + 10%FBS中以1-10×10 6个/ml的浓度培养CD34 +细胞,直到准备好转导它们。
  22. 在补充有450μg/ml Pip/Tazo的RPMI + 10%FBS中,将CD34-级分在37℃下以10×10 6 cfu/ml的浓度培养过夜。

转导CD34 +细胞

材料和试剂

  1. 在PBS中的RetroNectin(Clontech,目录号:T100A/B)20μg/ml(等分并冷冻1mg/ml -20℃的原液)
  2. Dulbecco's磷酸盐缓冲盐水(PBS)(Life Technologies,目录号:14190)
  3. FACS缓冲液(PBS中的4%FBS)
  4. 感染培养基 - 在Yseel的无血清T细胞培养基(Gemini,目录号:400-102)中的2%人血清白蛋白(美国红十字会,目录号:NDC 52769-451-05)(可选:补充50ng/ml人MGDF,SCF, 和Flt3-配体)
  5. 细胞因子培养基 - 用10%FBS,补充有100ng/ml人IL-3,IL-6,SCF的RPMI。
  6. 慢病毒载体,滴定
  7. 细胞刮刀

设备

  1. 生物安全柜
  2. 37& C孵育器
  3. 离心机
  4. 6孔板,未处理(BD Biosciences,Falcon ,目录号:351146)

程序

  1. 计算6孔组织培养板所需的转导孔的数目(1孔可以处理不超过8×10 6个细胞)。 用RetroNectin涂覆未处理的6孔板的所需数量的孔:将1.25ml的RetroNectin溶液(20μg/ml,在PBS中)分配到每个孔中,并且使覆盖的板在室温下在 清洁生物安全柜
  2. 从孔中除去RetroNectin溶液
  3. 向每个孔中加入1.25ml FACS缓冲液进行封闭。 让板在室温下放置30分钟。
  4. 取出FACS缓冲液,并用PBS洗孔一次
  5. 将PBS保留在涂层孔中,直到板准备使用。 您可以在4℃保存。 在即将接种细胞前从孔中取出PBS
  6. 对于转导效率对照,等分〜1×10 5个未转导的CD34 +细胞,并在96孔板中的200μl细胞因子培养基中在37℃下培养5-7天。
  7. 在RetroNectin包被的孔中(〜2×10 6个细胞/ml)在感染培养基中铺板剩余的CD34 +细胞,并在37℃下孵育1小时(参见下面步骤9中的替代方法) br />
  8. 将您的载体以2-10之间的感染复数(MOI)加入孔中。 轻轻混合并在37℃下孵育过夜
  9. 替代方法(对于较低效率的载体):首先向孔中加入感染培养基和载体,并在12℃下以3000×g离心90-120分钟,然后在12℃的培养基中添加细胞,轻轻旋转混合,在37℃下孵育过夜
  10. 第二天早上,收获细胞:轻轻刮擦孔底部,收集细胞,并计数
  11. 分装来自每种条件的〜1×10 5个细胞,并在96孔板中的200μl细胞因子培养基中在37℃下培养5-7天。 5-7天后,通过流式细胞术检查转导效率,例如,使用先前培养的未转导等分试样(参见步骤6)作为对照,观察记者。
  12. 对于将要移植的每只小鼠,将需要每个植入物0.5×10 6个转导的CD34 +细胞和用于注射的0.5×10 6个转导的CD34 +细胞。
  13. 对于植入的细胞,将每只小鼠的0.5×10 6个转导的CD34 +细胞与每个小鼠4.5×10 6个CD34 - 细胞组合,等分到无菌的1.5ml螺旋盖管中。旋转细胞沉淀它们,吸出上清液,再次旋转它们并吸出任何剩余的上清液,并将干燥的颗粒保持在冰上。
  14. 对于注射的细胞,每只小鼠自旋0.5×10 6个转导的CD34 +细胞以沉淀它们,吸出上清液,并将它们重悬于纯的RPMI中,每只小鼠150μl。 将它们保持在冰上。

组织移植构建NSG-CTL小鼠

材料和试剂

  1. 胎儿胸腺块
  2. CD34 +细胞用于植入和注射
  3. NSG小鼠,至少6-8周龄
  4. Matrigel,High Concentration(BD Biosciences,目录号:354248)
  5. 氯胺酮(Ketaject(Phoenix Pharmaceuticals,目录号:NDC 57319-542-02),100mg/ml)/甲苯噻嗪(Anaed注射液(Lloyd Laboratories,目录号:NADA 139-236),100mg/在PBS
  6. 异氟烷
  7. betadine
  8. 无菌盐水
  9. Carprofen(Rimadyl(Pfizer,目录号:NADA 141-199)),在无菌盐水中稀释1:100(在4℃下保持稀释长达24小时)
  10. PBS

设备

  1. 流式细胞仪
  2. 15ml锥形管
  3. 无菌纱布垫
  4. 60mm皿
  5. 15 ml锥形管
  6. 正位移移液管(1-20μl)和提示
  7. 带有40号刀片的Oster剪刀
  8. 60 mm培养皿
  9. 异丙醇擦拭
  10. 16号癌症植入针与圆形尖端(套管和套管针)
  11. 手术工具:针尖钳,弯钳,剪刀,止血钳,高压钳施放器和伤口夹
  12. Vicryl缝线,4-0(Owens& Minor,目录号:23000J304H)
  13. 胰岛素注射器
  14. 1 ml注射器

程序

  1. 手术前24小时,对小鼠使用2.7 Gy的剂量进行全身照射。
  2. 将胸腺片和培养基从烧瓶中倒入60mm皿中。将一些PBS倒入另一个盘子。
  3. 用PBS填充1ml注射器(无针)
  4. 冷却正位移移液管尖端通过将其放入开放的无菌管在冰中。与干燥的细胞团和Matrigel保持在冰上。重要的是保持Matrigel和任何管子或尖端,一直接触它冷,直到植入针装载。
  5. 麻醉小鼠:称量小鼠,并用15微升氯胺酮/赛拉嗪/克体重腹膜内注射。
  6. 当小鼠从氯胺酮/赛拉嗪变得缓慢时,将动物的鼻子放在15ml锥形管中,塞上浸有异氟烷的纱布,直到动物失去意识,其呼吸减慢到正常速率的约一半,并且不再响应爪爪。如果呼吸开始变得更快,在手术过程中保持该管手术以施用更多的异氟烷
  7. 打孔动物的耳朵编号或使用自己的首选识别方法
  8. 使用具有40号刀片的Oster剪刀,将每只小鼠的左侧从臀部到肩部刮在背部中心和腹部之间。
  9. 皮下注射0.3毫升稀释的Carprofen被动物的肩膀,然后将鼠标在无菌敷料的右侧,面向左边。
  10. 用PBS冲洗16号植入针的插管。
  11. 使用一对针尖镊子,将一块胸腺从盘中放入套管的开口,套管针正好在开口内,然后向后拉套管针以将组织吸入套管。
  12. 使用正位移移液管和冷却的尖端,将5微升冷基质胶放入一个管与干细胞沉淀,轻轻地搅拌足以使细胞悬浮。不要上下移动。当你慢慢拉回套管针以加载针头时,将基质胶/细胞悬液吸入套管的开口。在操作植入针时,使用移液器的助手更容易。
  13. 用Betadine擦拭鼠标的裸露皮肤,然后用异丙醇擦拭两次,擦拭该区域。
  14. 确定皮肤下最暗的点,其指示脾的位置。 肾脏是距离脾脏约5 mm。
  15. 用弯曲的钳子提起皮肤,并在肾脏平行于脾脏的皮肤上做出约15mm长的切口。 在下面的腹膜层做类似的切口
  16. 在雄性小鼠中,肾脏应该是可见的,并且可以通过按压在腹部上简单地挤出。 你可以用止血钳支撑肾脏。
  17. 在雌性小鼠中,卵巢阻断肾脏。 使用止血钳,拿起卵巢并拔出肾脏。
  18. 使用针尖镊子拔出肾小囊后端的一个小孔(1-2毫米)。
  19. 将植入针滑入这个孔并沿着肾脏,直到插管的开口被肾脏胶囊完全覆盖
  20. 轻轻挤出肾胶囊下的细胞和组织,然后将针头拉出。 胸腺片可以是粘的,所以你可以使用弯曲的镊子,以确保胸腺片不会与针一起出来。
  21. 用钳子提起腹膜,轻轻使用止血钳将肾脏推回原位
  22. 用双结在腹膜内扎一针。
  23. 使用两个高压锅关闭皮肤。
  24. 混合保存用于注射的转导的CD34 +细胞,并吸入150μl(0.5×10 6个细胞)进入胰岛素注射器。 通过眶后静脉注射将这些细胞注入小鼠
  25. 将一滴PBS放在每只眼睛上,将鼠标放回笼子里。
  26. 当所有的小鼠都被植入后,确认动物在离开前恢复意识
  27. 术后护理:手术后的第二天,向每只小鼠皮下注射0.3ml稀释的Carprofen和1.2ml无菌盐水。在手术后第2和3天,向每只小鼠皮下注射1.5ml无菌盐水。手术后监测小鼠和切口10天。取出高压灭菌器,10天后称重小鼠。
  28. 在8-10周后,通过使小鼠出血并对外周血进行FACS分析来检查移植,标记物例如CD45,CD3,CD4,CD8和载体应该表达的任何基因的染色。图2中提供了典型的流式细胞术图,即人CD3(T细胞标记)和转基因的HIV特异性TCR的染色。

    图2.  外周血中的TCR +细胞样本

  1. 有关类似类型的移植程序的视觉参考,请参阅2012年可视化实验杂志中的Vatakis等人。
    http: //www.jove.com/video/4181/using-blt-humanized-mouse-as-stem-cell-based-gene-therapy-tumor )。

变异

  1. 转导的CD34 +细胞可以可行地冷冻而不是手术后注射。 在这种情况下,不要在手术前照射小鼠。 相反,等待4-6周,解冻细胞,照射小鼠(2.70Gy),并以0.5x10 6细胞/小鼠的轨道注射细胞。 这些小鼠的术后护理在手术后第一天将为0.3ml卡洛芬。 不需要注射盐水。

致谢

这项工作由来自NIH(编号R01AI078806(SGK),加利福尼亚艾滋病毒/艾滋病研究计划(CHRP)(编号163893)(SGK),UC Multicampus研究计划和来自加利福尼亚抗病毒药物发现中心编号MRPI-143226),以及由加州大学洛杉矶分校艾滋病研究中心(CFAR)资助(编号P30 AI28697)。

参考文献

  1. Kitchen,S.G.,B.R.Levin,G.Bristol,V.Rezek,S.Kim,C.Aggilera-Sandoval,A.Balamurugan,O.O.Yang和J.A.Zack(2012)。 体内抑制来自工程造血的抗原特异性T细胞的HIV干细胞。 PLoS Pathog 8(4):e1002649。
  2. Melkus,M.W.,J.D.Estes,A.Paddgett-Thomas,J.Gatlin,P.W.Denton,F.A.Othieno,A.K.Wege,A.T.Haase和J.V.Garcia(2006)。 人性化小鼠对EBV和TSST-1具有特异性适应性和先天免疫反应。 em> Nat Med 12(11):1316-1322。 
  3. Vatakis,D.N.,Bristol,G.C.,Kim,S.G.,Levin,B.,Liu,W.,Radu,C.G.,Kitchen,S.G.and Zack,J.A。(2012)。 使用BLT人源化小鼠作为基于干细胞的基因治疗肿瘤模型 J Vis Exp (70):e4181。


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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Levin, B., Bristol, G., Galic, Z. and Kitchen, S. G. (2013). Construction of NSG-CTL Mice. Bio-protocol 3(4): e329. DOI: 10.21769/BioProtoc.329.
  2. Kitchen, S. G., B. R. Levin, G. Bristol, V. Rezek, S. Kim, C. Aguilera-Sandoval, A. Balamurugan, O. O. Yang and J. A. Zack (2012). In vivo suppression of HIV by antigen specific T cells derived from engineered hematopoietic stem cells. PLoS Pathog 8(4): e1002649.
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