MM1S-luc cells were harvested and injected on day 0 at 2 × 106 cells per mouse intravenous (i.v.) into 6–9-week-old male NSG mice (NOD.Cg-PrkdcSCIDIl2rgTM1wJ1/SzJ, Jackson labs). Tumor growth was monitored during the study using bioluminescence imaging (BLI). BLI measurement and randomization into treatment groups (n=4 per group) was performed on day 6. On days 7, 14, 21 and 28, CAR T-cells were thawed from cryovials, washed, and resuspended for administration via i.v. injection with vehicle, mock-transfected CD8+ or Descartes-08 cells (20 × 106 cells per mouse). Pre-conditioning of mice was performed on days 13, 20, and 27 by administration of cyclophosphamide at 1.2 mg/mouse via intraperitoneal (i.p.) injection [29 (link), 30 (link)]. Descartes-08 cells were further engineered to reduce expression of T cell receptor (TCR) by CRISPR/Cas9 deletion of the Tcra gene (with ~95% knockout efficiency) to inhibit non-specific xenogeneic and allogeneic responses with repeat-dose CAR T-cell infusions [31 (link)]. Animals were monitored twice daily. Animals showing obvious signs of distress or pain including inability to drink or feed or a >20% reduction in body weight were humanely euthanized prior to study end according to Institutional Animal Care and Use Committee-approved protocol.
Nod cg prkdcscidil2rgtm1wj1 szj
Manufactured by Jackson ImmunoResearch
Sourced in United States
NOD.Cg-PrkdcscidIl2rgtm1Wj1/SzJ is a mouse strain that is deficient in T and B cells, as well as natural killer cells, due to mutations in the Prkdc and Il2rg genes. This strain is commonly used in research as a model for studying immune system function and development.
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9 protocols using nod cg prkdcscidil2rgtm1wj1 szj
1
Targeting Solid Tumors with Engineered CAR T Cells
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Patient-Derived Xenograft Tumor Model
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Animal research was approved by the UW-Madison Institutional Animal Care and Use Committee. Organoids from Patient PC13 were pelleted, re-suspended in media, and mixed 1:1 with Matrigel. This mixture was subsequently injected (100 μl) subcutaneously into bilateral flanks of female NOD scid gamma mice at 6 weeks old (NOD.Cg-PrkdcscidIl2rgtm1Wj1/SzJ, The Jackson Laboratory) for initial patient-derived xenograft (PDX) establishment. For treatment experiments, extracted tumors were mechanically minced to form a cell suspension, which was then mixed with Matrigel for injection into experimentally naïve female athymic nude mice at 6 weeks old (Hsd:Athymic nude-Foxn1nu/nu, Envigo). Tumor volume was measured with calipers using the formula 0.5 * length * width2. When average tumor volume reached ~150 mm3, mice were randomized into two groups. Twenty mice received 100 mg/kg gemcitabine and 100 mg/kg nab-paclitaxel weekly via intraperitoneal injection while 23 control mice received only PBS weekly. Tumor volume was measured twice weekly. Mice were euthanized and tumors were collected when humane endpoints were reached.
3
Tracking Metastasis in NSG Mice
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This study was approved by the Institutional Animal Care and Utilization Committee of the American University of Beirut (IACUCC#10-07-154). Immune-deficient NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wj1/SzJ, Jackson Laboratory, Bar Harbor, ME, USA) were injected with 2 × 106 MDA-MB-231 cells into the subcutaneous area of the neck region. Cells were washed twice with PBS, re-suspended in serum-free media and passed through a 40-μm cell strainer to remove any cell clumps before injection. This model induces growth of primary tumor at the injection site, which would subsequently metastasize to secondary sites, as described previously [18 (link)]. Experimental groups (20 mice per group) included mice injected with parental MDA-MB-231 cells (control), shCx43 cells or Cx43D cells. Subsets of mice from each group were used for primary tumor analysis (onset and tumor volume), survival studies and metastasis studies at week 9 post-grafting using molecular analysis and histological examination.
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Murine Models for Immunology Research
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Wild‐type (WT) C57BL/6 J (BL6) and NOD.Cg‐PrkdcscidIl2rgtm1Wj1/SzJ [nonobese diabetic/severe combined immunodeficiency (NOD/SCID)/IL2Rγ KO, NSG] mice were purchased from Jackson Laboratory, maintained under specific pathogen‐free conditions and given food and water ad libitum. Age‐ and sex‐matched mice at least 8 weeks old were used. All studies were approved by our Institutional Animal Care and Use Committee.
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Targeting Solid Tumors with Engineered CAR T Cells
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MM1S-luc cells were harvested and injected on day 0 at 2 × 106 cells per mouse intravenous (i.v.) into 6–9-week-old male NSG mice (NOD.Cg-PrkdcSCIDIl2rgTM1wJ1/SzJ, Jackson labs). Tumor growth was monitored during the study using bioluminescence imaging (BLI). BLI measurement and randomization into treatment groups (n=4 per group) was performed on day 6. On days 7, 14, 21 and 28, CAR T-cells were thawed from cryovials, washed, and resuspended for administration via i.v. injection with vehicle, mock-transfected CD8+ or Descartes-08 cells (20 × 106 cells per mouse). Pre-conditioning of mice was performed on days 13, 20, and 27 by administration of cyclophosphamide at 1.2 mg/mouse via intraperitoneal (i.p.) injection [29 (link), 30 (link)]. Descartes-08 cells were further engineered to reduce expression of T cell receptor (TCR) by CRISPR/Cas9 deletion of the Tcra gene (with ~95% knockout efficiency) to inhibit non-specific xenogeneic and allogeneic responses with repeat-dose CAR T-cell infusions [31 (link)]. Animals were monitored twice daily. Animals showing obvious signs of distress or pain including inability to drink or feed or a >20% reduction in body weight were humanely euthanized prior to study end according to Institutional Animal Care and Use Committee-approved protocol.
6
Mouse Strain Maintenance and Utilization
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Male and female wild-type (WT) C57BL/6J (BL6) and NOD.Cg-PrkdcscidIl2rgtm1Wj1/SzJ (non-obese diabetic/severe combined immunodeficiency (NOD/SCID)/interleukin-2Rγ KO, NSG) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA), maintained under specific pathogen-free conditions and given food and water ad libitum. Eight- to ten-week-old age- and sex-matched mice were used for experiments. All animal studies were approved by our Institutional Animal Care and Use Committee.
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NOD SCID Gamma Mice for Research
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In Vivo Tumor Growth Inhibition
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Example 3
These protocols were used in many of the experiments of the figures. Tumor cells were implanted subcutaneous (SC) in the right flank of NSG (NOD.Cg-Prkdcscid Il2rgtm1Wj1/SzJ) mice (The Jackson Laboratory, Cat. No. 005557) and allowed to grow until an established tumor with a mean volume of around 200 mm3 was reached. In parallel human T cells were cultured in T cell media (X-VIVO 15 [Lonza, Cat. No. 04-418Q], 5% Human Serum, 1% Penicillin/Streptomycin, 0.01 mM 2-Mercaptoethanol) in a G-Rex100M gas permeable flask (Wilson Wolf Cat. No. 81100S) with MACSiBeads from the T Cell Activation/Expansion Kit (Miltenyi Cat. No. 130-091-441) for around 10 days and supplemented with recombinant human IL-2 protein. Tumor growth in mice and human T cell activation/expansion were coordinated so that on Day 0 of the study mice were randomized into groups (N=6) based on tumor size; each were then injected intravenous (IV) with 2.5×106 cultured human T cells and administered the first dose of the COBRA or control molecules. Mice were dosed every 3 days for 7 doses (Days 0, 3, 6, 9, 12, 15 and 18) and then followed for an additional 2-3 weeks until tumors reached >2000 mm3 in volume or the study was terminated. Tumor volumes were measured every 3 days.
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Syngeneic Mouse Models in Cancer Research
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