Name: derivation of thymic lymphoma t-cell lines from atm-/- and p53-/- mice
Uploaded: 2011-04-03T14:00:00
Description: Watch this Scientific Journal Video about Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice at JoVE.com

作者衷心感谢鲍里斯Reizis在哥伦比亚大学的微生物学和免疫学系博士的有益讨论。我们也希望在康奈尔大学兽医学院，感谢冯富珍博士Bynoe，罗德曼Getchell博士和Deequa Mahamed从微生物学和免疫学系与流式细胞仪和视频编辑的协助下，黄路为技术援助。作者还希望感谢魏斯实验室杜哈明和魏斯实验室的成员，他们的手稿和视频制作，严格审查和斯蒂芬妮Yazinski育种和基因分型p53基因- / -小鼠。在康奈尔大学机构动物护理和使用委员会规定的准则和法规的规定进行动物实验。这项研究是由美国农业部部，合作国家研究，教育和推广服务，动物健康和疾病研究计划（GED和RSW）和美国国立卫生研究院拨款R03 HD058220和R01CA108773（RSW）提供的资金支持部分。视频制作杜哈明魏斯实验室的人员使用的内部设施。

1。肿瘤剖析<ol><li>肿瘤清扫干净的纸巾或一次性手术悬垂放在解剖台上，喷以70％的乙醇。 </li><li>本议定书小鼠常规安乐死的CO 2。安乐死鼠标放置在解剖台上，和动物两侧喷用乙醇。鼠标是在地方上的夹层板，使用四个或更多的推引脚通过插入腿和喷用70％乙醇彻底。 </li><li>从耻骨头的基地使用无菌剪刀，同时也避免了潜在的肌肉和渗透到体腔，在中线的皮肤被切断。 </li><li>打开腹腔...

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	<li>Barlow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atm-deficient+mice:+a+paradigm+of+ataxia+telangiectasia.">Atm-deficient mice: a paradigm of ataxia telangiectasia.</a> Cell. 86, 159-171 (1996).</li><li>Elson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+defects+in+ataxia-telangiectasia+protein-deficient+mice.">Pleiotropic defects in ataxia-telangiectasia protein-deficient mice.</a> Proc Natl Acad Sci U S A. 93, 13084-13089 (1996).</li><li>Kuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Atm-/-+thymic+lymphoma+cell+proliferation+in+vitro+and+in+vivo+by+dexamethasone.">Control of Atm-/- thymic lymphoma cell proliferation in vitro and in vivo by dexamethasone.</a> Cancer Chemother Pharmacol. 55, 203-212 (2005).</li><li>Browne, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+a+catalytic+antioxidant+corrects+the+neurobehavioral+defect+in+ataxia-telangiectasia+mice.">Treatment with a catalytic antioxidant corrects the neurobehavioral defect in ataxia-telangiectasia mice.</a> Free Radic Biol Med. 36, 938-942 (2004).</li><li>Jacks, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+spectrum+analysis+in+p53-mutant+mice.">Tumor spectrum analysis in p53-mutant mice.</a> Curr Biol. 4, 1-7 (1994).</li><li>Chervinsky, D. S., Lam, D. H., Zhao, X. F., Melman, M. P., Aplan, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+T+cell+leukemia+cell+lines+established+from+SCL/LMO1+double+transgenic+mice.">Development and characterization of T cell leukemia cell lines established from SCL/LMO1 double transgenic mice.</a> Leukemia. 15, 141-147 (2001).</li><li>Sharma, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1+contributes+to+mouse+T-cell+leukemia+by+directly+inducing+the+expression+of+c-myc.">Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc.</a> Mol Cell Biol. 26, 8022-8031 (2006).</li><li>Maser, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomally+unstable+mouse+tumours+have+genomic+alterations+similar+to+diverse+human+cancers.">Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers.</a> Nature. 447, 966-971 (2007).</li></ol>

在这个协议中，我们提供了详细的程序，建立从两个不同的鼠标基因型的小鼠的T 细胞系;自动取款机- / - [FVB/N- ATM tm1Led / A]和p53 - / - [129/S6- Trp53 tm1Tyj / J]。 。通过使用这个协议中，共有6（满分7次尝试）自动取款机- / -三（5次尝试）P53 - / -小鼠的T细胞系已经建立在我们的实验室。 / - -...

<ul><li>冰桶</li><li> 70％的乙醇</li><li>覆盖铝箔和引脚的泡沫块组成的夹层板</li><li>两套小无菌剪刀和镊子</li><li>无菌150毫米2培养皿（赛默飞世尔科技，沃尔瑟姆，马，猫＃08-757-13） </li><li>无菌18表针</li><li> 50 ml锥形管（康宁公司，康宁纽约猫＃430829） </li><li> 10％的福尔马林</li><li>组织培养瓶<ul><li> T - 25（TPP，Trasadingen，瑞士，CAT＃90025） </li><li> T - 75（TPP，Trasadingen，瑞士，CAT＃90075） </li></ul></li><li> 6孔板（康宁公司康宁公司，纽约;猫＃3506） </li><li>无菌磷酸盐缓冲液pH值7.2（PBS） </li><li>培养基<ul><li> RPMI 1640培养液，与25毫米的HEPES，200毫米以下补充L -谷氨酰胺（龙沙，Walkersville，MD;猫＃12 - 115F）： <ul><li> 10％热灭活（56℃，30分钟）小牛血清（FBS，Hyclone公司，UT斯达康，洛根＃SH30088.03猫） </li><li> 1％青霉素和链霉素（Mediatech，弗吉尼亚州马纳萨斯的猫＃30 - 002 - CI） </li><li> 1％的不重要的氨基酸（猫＃25 - 025 - CI Mediatech） </li><li> 55毫米2β-巯基乙醇（Sigma公司，圣路易斯，密苏里州，猫＃M752） </li></ul></li></ul></li><li>二甲基亚砜（DMSO; Calbiochem，拉霍亚，CA;猫＃317275） </li><li>抗体（eBioscience公司，加利福尼亚州圣迭戈） <ul><li>抗CD3 - FITC（货号＃11-0031-85） </li><li>抗CD4 - APC（货号＃17-0041-83） </li><li>抗- CD8 - PE（货号＃12-081-85） </li></ul></li><li>牛血清白蛋白（BSA;生命科技，大岛，NY;猫＃11018-017） </li><li>伴刀豆球蛋白A（Sigma公司猫＃C5275） </li><li> 1.8毫升冷冻瓶（NUNC，丹麦的罗斯基勒，猫＃368632） </li></ul>

derivation of thymic lymphoma t-cell lines from atm-/- and p53-/- mice

建立的细胞系是一个重要的的研究工具，它可以减少使用实验动物的研究。某些菌株的转基因小鼠， 如 ATM - / - 和 p53 - / -胸腺淋巴瘤不断发展早在生活中1,2，因此，可以作为一个可靠的来源小鼠T细胞株的推导。在这里，我们建立了小鼠胸腺淋巴瘤的T细胞系的发展，而不需要添加白细胞介素1,3以前的协议来描述一个详细的协议。从年龄在3至6个月的小鼠肿瘤收获，最早表现的基础上，弯腰驼背的姿势观察可见肿瘤，呼吸困难，穷人疏导，并在敏感品系1,4浪费。我们已经成功地建立了几个使用此协议的T细胞系和近交系的自动取款机- / - [FVB/N- 自动柜员机tm1Led / J] 2 和p53 - / - [129/S6- Trp53 tm1Tyj / J] 5只。我们进一步表明，既定的T细胞的人口超过90％表示CD3，CD4和CD8。与稳定建立的细胞株，使用本议定书所产生的T细胞已传了一年多。

Watch this Scientific Journal Video about Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice at JoVE.com

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

在这个视频中，我们展示了一个协议来建立小鼠胸腺淋巴瘤细胞株。通过这个协议，我们已经成功地建立了几个从ATM / T细胞线 - 和p53 - / - 小鼠胸腺淋巴瘤。

胸腺淋巴瘤的T细胞系的推导 ATM - / -</sup P53 - / -</sup鼠标

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

Research

JoVE Journal

Immunology and Infection

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

与预先定义的T细胞受体特异性Transnuclear小鼠对弓形虫获得通过体细胞核移植

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

科研

免疫与感染

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

考试流式细胞仪检测胸腺阳性和阴性选择

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

对T细胞推导<em&gt;在体外</em&gt;从小鼠胚胎干细胞

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Mouse NaÃƒÆ’Ã‚Â¯ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Preparation and Applications of Organotypic Thymic Slice Cultures

制备及胸腺器官切片文化中的应用

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

使用成像流式细胞仪T细胞同种异体反应性测定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

胸腺淋巴瘤的T细胞系的推导 ATM - / - P53 - / -鼠标

胸腺淋巴瘤的T细胞系的推导 ATM^{- / - P53^{- / -鼠标}}