B6.129p2 cxcr3tm1dgen j

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B6.129P2-Cxcr3tm1Dgen/J is a strain of genetically modified mouse. This mouse expresses a targeted mutation in the Cxcr3 gene, which encodes the Cxcr3 chemokine receptor. The mutation was introduced using gene-targeting techniques.

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9 protocols using b6.129p2 cxcr3tm1dgen j

Genetically Modified Mouse Models for Tumor Studies

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6- to 8-week-old female C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD). IFN-γ receptor 1-deficient (ifngr1^tm1Agt) mice (IFNγR−/−) and CXCR3-deficient (B6.129P2-Cxcr3^tm1Dgen/J) mice (CXCR3−/−) have been previously described (Jackson Laboratories) (35 (link)). Mice were housed in the oncology animal facility of the Johns Hopkins Hospital (Baltimore, MD). All animal procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals.
The tumor cell line used in our study was TC-1. TC-1 cells were subjected to RapidMAP (Taconic Farms, Rensselaer, NY) testing, a panel of PCR tests for rodent viruses, most recently in May 2011 with negative results. In order to trace the tumor growth in vivo, TC-1 transfected with Luciferase (TC-1-Luc) was generated by lentivirus stable transfection as previously described (36 (link)). Cells were cultured in RPMI1640 medium containing 10% FBS, 2mM L-glutamine, 10% sodium pyruvate, 10% non-essential amino acids, and 100pg/ml streptomycin in a humidified atmosphere of 5% CO₂/95% air at 37°C.

Soong R.S., Song L., Trieu J., Knoff J., He L., Tsai Y.C., Huh W., Chang Y.N., Cheng W.F., Roden R.B., Wu T.C, & Hung C.F. (2014). Toll like receptor agonist imiquimod facilitates antigen-specific CD8+ T cell accumulation in the genital tract leading to tumor control through interferon-γ. Clinical cancer research : an official journal of the American Association for Cancer Research, 20(21), 5456-5467.

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Murine Models for Cancer Research

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We purchased Balb/c (#000651), C57BL/6 (#000664), B6.129P2-Cxcr3^tm1Dgen/J (005796), and Thy1.1 (#005443) mice from The Jackson Laboratory. FcƴRIIB^-/- mice (#Fcgr2b-Model 579) were purchased from Taconic. NOD.CB17-Prkdc^scid/J (SCID) mice were a gift from Dr. Richard Gorlick (The University of Texas MD Anderson Cancer Center)^{42 (link)}. Both female and male mice were used for all experiments. All mice were aged 5 to 8 weeks when the experimental procedures began. All animals were housed in a specific pathogen-free room with a 12-h light/dark cycle with free access to a standard rodent diet and water at ambient temperature maintained between 18 and 23 °C and humidity between 40 and 60%. We performed all animal experiments in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) at MD Anderson.

Zhao Q., Hu J., Kong L., Jiang S., Tian X., Wang J., Hashizume R., Jia Z., Fowlkes N.W., Yan J., Xia X., Yi S.F., Dao L.H., Masopust D., Heimberger A.B, & Li S. (2023). FGL2-targeting T cells exhibit antitumor effects on glioblastoma and recruit tumor-specific brain-resident memory T cells. Nature Communications, 14, 735.

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Cuprizone-Induced Demyelination in Mice

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Toxic demyelination was induced in 8-week-old wild type C57BL/6 (WT, Charles River) and CXCR3-deficient mice (CXCR3-/-; B6.129P2-Cxcr3tm1Dgen/J, Jackson Laboratory) fed with 0.2% (w/w) cuprizone (bis-cyclohexanone oxaldihydrazone, Sigma-Aldrich, München, Germany). cuprizone was mixed into a powdered ground standard rodent chow. Before cuprizone administration, all mice of the study were adapted to powdered normal chow for one week. cuprizone diet was then maintained for five weeks following four additional days on normal chow, representing the stage of CNS-remyelination. As a marker for the clinical outcome of cuprizone administration, the body weight of all mice was monitored twice a week. A total of 4 to 5 WT and CXCR3-/- mice were analyzed at each of the four time points of the experiment (0, 3, 5, and 5 weeks + 4 additional days normal chow/ 5.5 weeks). Animals were deeply anesthetized and transcardially perfused with ice cold phosphate buffered saline (PBS). Brains were removed immediately and cut along the sagittal midline. Brain hemispheres were fixed in 4% paraformaldehyde for 12 hours following paraffin-embedding. The remaining hemisphere was further processed for flow cytometry and quantitative PCR analysis.

Krauthausen M., Saxe S., Zimmermann J., Emrich M., Heneka M.T, & Müller M. (2014). CXCR3 modulates glial accumulation and activation in cuprizone-induced demyelination of the central nervous system. Journal of Neuroinflammation, 11, 109.

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Transgenic Mouse Models for Immune Research

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B6.Cg-Rptor^tm1.1Dmsa/J (B6 Raptor-flox), Rictor^tm1.1Klg/SjmJ (B6 Rictor-flox), B6.Cg-Tg (CD2-icre) 4Kio/J (hCD2-iCre), B6.129S7-Ifngtm1Ts/J (IFN-γ^-/-), C57BL/6-Il17atm1Bcgen/J (IL-17A^-/-), B6.129S4-Ifngtm3.1Lky/J (IFN-γ-eYFP), C57BL/6-Il17atm1Bcgen/J (IL-17-GFP), B6.129P2-Cxcr3tm1Dgen/J (CXCR3^-/-), B6.129S6-Tbx21tm1Glm/J (Tbx21^-/-) and B6.129P2-Tcrd^tm1Mom/J (TCR δ^−/−) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). CD45.1 mice were given by Zhongjun Dong from Tsinghua University (Beijing, China). Sex- and age-matched animals were randomly assigned to different groups. All mice were maintained under SPF conditions and all animal procedures were approved by the Institutional Animal Care and Use Committee of Jinan University.

Liu Q., Yang Q., Wu Z., Chen Y., Xu M., Zhang H., Zhao J., Liu Z., Guan Z., Luo J., Li Z.Y., Sun G., Wen Q., Xu Y., Li Z., Chen K., Ben X., He W., Li X., Yin Z., Hao J, & Lu L. (2022). IL-1β-activated mTORC2 promotes accumulation of IFN-γ+ γδ T cells by upregulating CXCR3 to restrict hepatic fibrosis. Cell Death & Disease, 13(4), 289.

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Tumor growth in C57BL/6 mouse model

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Female 5–6 weeks old wild type (WT) C57BL/6 (B6) mice (H-2b) and CXCR3 knock out (KO) mice (B6.129P2-CXCR3^tm1/Dgen/J) were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were raised in a specific pathogen-free environment under a temperature and light-controlled room with free access to food and water. Animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (Protocol No. 18012053). Anesthesia was conducted with isoflurane (Piramal Critical Care, PA, USA). B6 mice were injected with 1×10⁷ Hepa1–6 cells subcutaneously in the left flank containing 100 μl growth factor depleted Matrigel (CORNING, MA, USA). Tumor size was calculated every 3 days by caliper. Tumor volume=L×W× ((L+W)/2). Body weight was recorded every 3 days. After 2 weeks, mice were sacrificed under anesthesia by cervical dislocation. Tumors were harvested for single cell suspension for flow cytometry or frozen with OCT, and 5 μM sections were cut.

Yan Y., Zheng L., Du Q., Yazdani H., Dong K., Guo Y, & Geller D.A. (2021). Interferon Regulatory Factor 1(IRF-1) activates anti-tumor immunity via CXCL10/CXCR3 axis in Hepatocellular Carcinoma (HCC). Cancer letters, 506, 95-106.

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Genetically Modified Mouse Strains

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C57BL/6J, B6.PL-Thy1^a/CyJ (CD90.1), B6.SJL-Ptprc^a Pepc^b/BoyJ (CD45.1), B6.129S2-Ighm^tm1Cgn/J (µMT), B6.129S7-Ifng^tm1Ts/J (IFNγ^−/−), B6.129S7-Ifngr1^tm1Agt/J (IFNγR^−/−), B6.129P2-Ccr5^tm1kuz/J (CCR5^−/−), and B6.129P2-Cxcr3^tm1Dgen/J (CXCR3^−/−) were purchased from The Jackson Laboratory (Bar Harbor, ME). NR1 transgenic mice expressing a TCR transgene specific for the C. trachomatis antigen Cta1_133–152 have been described previously (25 (link)). CXCR3^−/−CCR5^−/− mice were generated by crossing CXCR3^−/− and CCR5^−/− mice. Mice were maintained within the Harvard Medical School Center for Animal Resources and Comparative Medicine. All experiments in this report were approved by Harvard’s Institutional Animal Care and Use Committee.

Nogueira C.V., Zhang X., Giovannone N., Sennott E.L, & Starnbach M.N. (2015). Protective immunity against Chlamydia trachomatis can engage both CD4+ and CD8+ T cells and bridge the respiratory and genital mucosae. Journal of immunology (Baltimore, Md. : 1950), 194(5), 2319-2329.

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Fibroblast Isolation and Cytokine Stimulation

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NIH-3T3 fibroblasts were obtained from European Collection of Cell Cultures (ECACC) and cultured in Dulbecco's Modified Eagle Medium (Gibco/Invitrogen, Ireland) supplemented with 10% heat inactivated FBS, (Sigma Aldrich, Ireland), 5mM L-glutamine (Gibco/Invitrogen) penicillin (100U/ml), and streptomycin (100μg/ml) (Gibco/Invitrogen) at 37° Celsius in humidified 5% CO2. Primary lung fibroblasts were isolated from CXCR3 -/-mice (B6.129P2-Cxcr3tm1Dgen/J, Jackson laboratories) or wild type C57BL/6 mice (Charles River, UK.) as previously described (3) . Primary fibroblasts were used between passages 2-8 for all experiments and cultured in standard media conditions, as above. To prepare samples for analysis, cells were serum starved for 18h then incubated in medium containing vehicle alone or supplemented with IL-13 (Biolegend) CXCL9, CXCL10 (Biolegend) at the time periods indicated. To investigate CXCR3 dependent signalling cells were pre-treated with 500nM of CXCR3 antagonist 500586 (Calbiochem) for one hour before stimulation with cytokines/chemokines.

Worrell J.C., Walsh S.M., Fabre A., Kane R., Hinz B, & Keane M.P. (2020). CXCR3A promotes the secretion of the antifibrotic decoy receptor sIL-13Rα2 by pulmonary fibroblasts. American journal of physiology. Cell physiology, 319(6).

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Mice Strain Comparison for Protozoan Infection

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C57BL/6J (WT) mice were purchased from Clea Japan (Tokyo, Japan). CXCR3KO mice (B6.129P2-Cxcr3tm1Dgen/J, Stock Number: 005796) were purchased from the Jackson Laboratory (Bar Harbor, ME). The CXCR3KO mice have been backcrossed to C57BL/6 at least six times before being imported to our laboratory. All animals were housed in cages (<6 mice/cage, 225 mm × 340 mm × 155 mm) containing wood chip bedding under specific-pathogen-free conditions in the animal facility of the National Research Center for Protozoan Diseases at Obihiro University of Agriculture and Veterinary Medicine. Male mice at 9–11 weeks old were used for in vivo experimental infection and preparation of peritoneal macrophages. The mean ± SD starting weight was 23.6 ± 1.0 g for WT mice and 23.7 ± 1.3 g for CXCR3KO mice. Mice at 6–8 weeks old were used as the parents of fetal mice for the preparation of primary glial cells.

Umeda K., Goto Y., Watanabe K., Ushio N., Fereig R.M., Ihara F., Tanaka S., Suzuki Y, & Nishikawa Y. (2021). Transcriptomic Analysis of the Effects of Chemokine Receptor CXCR3 Deficiency on Immune Responses in the Mouse Brain during Toxoplasma gondii Infection. Microorganisms, 9(11), 2340.

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Isolation and Culture of Cortical and Hippocampal Neurons

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Cortical and hippocampal neurons from P0 to P1 WT or KO mouse pups (B6.129P2-Cxcr3tm1Dgen/J; the Jackson Laboratory, strain #005796) were obtained as described previously (118 (link)), with minor modifications. Briefly, papain-dissociated cells were filtered through a 0.4-μm cell strainer (Corning, #431750) to enrich for neurons, centrifuged at 500g for 5 min to eliminate small debris, and suspended in complete primary neuronal medium consisting of B-27 Plus Neuronal Culture system (Thermo Fisher Scientific, #A3653401) and 1× GlutaMAX (Thermo Fisher Scientific, #35050061) without antibiotics. Cells were seeded at 50,000 to 150,000 live cells/cm² into plates coated with poly-d-lysine (0.01%, w/v; Sigma-Aldrich, P6407). Medium was fully exchanged 1 day after plating (DIV 1) with subsequent half-medium exchanges every 3 to 4 days.

Licht-Murava A., Meadows S.M., Palaguachi F., Song S.C., Jackvony S., Bram Y., Zhou C., Schwartz R.E., Froemke R.C., Orr A.L, & Orr A.G. (2023). Astrocytic TDP-43 dysregulation impairs memory by modulating antiviral pathways and interferon-inducible chemokines. Science Advances, 9(16), eade1282.

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