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，用无菌剪刀和镊子，小心不要损坏内部器官的一双新。 </li><li>通过解剖和偏转左肋揭示不损害血管的肿瘤，打开胸腔。 </li><li>从其他器官中分离出来的肿瘤，转移到一块冷PBS总的肿瘤约40-50％（约1.5 × 0.8 × 0.8厘米）保持在冰上。 </li><li>肿瘤的剩余部分可以保存为其他实验可以在这个时候进行一个完整的尸体剖检。 </li></ol> 2。培养淋巴瘤细胞<ol><li>喷雾管中，用70％乙醇和转移到了一个生物安全柜的解剖肿瘤。 </li><li> 10毫升培养液中添加150毫米无菌培养皿。 </li><li>转移到培养皿中，用吸管轻轻吸一块肿瘤。 </li><li>弯曲两个无菌18号针头面临斜角方。 </li><li>游离的肿瘤，使用弯曲的针。 </li><li>涡流的菜，请点击轻轻地放在一个角度，一个侧面收集细胞悬液，同时避免了大块肿瘤。 </li><li>吸50 mL锥形管的细胞悬液和转让，同时避免了大块肿瘤。 </li><li>洗净同一管板与10毫升的媒介和转移，同时避免大块肿瘤。 </li><li>混合轻轻的淋巴细胞和基质细胞组成的细胞悬液，并转移到了T - 25的组织培养瓶和10毫升到5毫升的T - 75的组织培养瓶中。轻轻漩涡的烧瓶。 </li><li>使用剩余的细胞悬浮于2.5毫升的最终体积（在使用几种类型的文化船只不同密度的培养细胞最大限度地建立一个T -细胞株的可能性）2，4，10100和1000倍稀释。 </li><li>板2.0毫升每稀释到6孔板个别井。轻轻旋流板。 </li><li>在加湿空气中的CO 2气氛中孵育48 h后的细胞。 </li></ol> 3。饲养淋巴瘤细胞<ol><li>孵化48小时后，添加5毫升的中型的T - 75烧瓶烧瓶的T - 25和10毫升介质沿墙，干扰最小的细胞。 </li><li>加入2毫升培养基，每孔的墙沿，干扰最小的细胞的6孔板。 </li><li>孵育细胞额外的72小时</li></ol> 4。传代淋巴瘤细胞<ol><li>初步通过<ol><li>在孵化第五天结束时，在1:1的比例传代细胞，同时保留原瓶和板（约3x106 /毫升的浓度保持在第一段落的细胞密度，促进建立一个成功的连续细胞系）。 <ol class="lalpha"><li>的T - 25烧瓶，转移到一个新的中等5毫升容量瓶中含有5 ml的细胞悬液。添加到原瓶5毫升的中型。 </li><li>的T - 75烧瓶中，转移到一个新的含有10毫升中等的容量瓶的10 ml的细胞悬液。添加到原瓶10毫升介质。 </li><li>对于6孔板，每口井从传输到相应的一个新的含有2毫升培养基板，每孔井2 ml的细胞悬液。添加到每一个原来的水井以及2毫升培养基。 </li></ol></li><li>所有额外的72小时孵育瓶和盘子根据我们的经验，大多数细胞系建立了从这些通道之一的文化（松散贴壁细胞悬浮聚合的附着的基质细胞的存在是一个已建立的细胞株的早期迹​​象）。 </li><li>对于较少或没有松散贴壁细胞悬浮聚合的文化，饲料仔细中期每72小时更换约50％的烧瓶或板为了避免去除细胞的原始文化，文化的船只罚站和不受干扰至少5分钟，让在底部的上清液中期之前温柔的愿望重力细胞泥沙。 </li></ol></li><li>维护建立的细胞株<ol><li>建立的细胞株传代成新瓶和盘子。对于T - 25和T - 75烧瓶中，加入5毫升的原件或通过暂停到5毫升中等。对于6孔板中，加入7 mL的容量瓶中的T - 25中型3毫升的原件或通过一个细胞悬液。饲料和更换新鲜培养液中删除的卷保留原或通过一种文化。 </li><li>通道设立了​​新的T - 25培养瓶细胞株。细胞系有一个倍增时间为18-24小时。文化是经常保持在1 - 3x106 /毫升的密度，每3天传代。以前通过文化需要的量添加到新的介质，以弥补10毫升。饲料和保留了原有的文化。 </li><li>几个段落后，非贴壁细胞会自我维持不贴壁的基质细胞的贴壁细胞数量较少，每个通道进行。除了这一点，可以保持文化的非组织培养治疗的T - 25培养瓶。 </li></ol></li></ol> 5。冻结和恢复淋巴瘤细胞株<ol><li>新鲜配制RPMI 1640培养液含20％胎牛血清和10％DMSO的培养基中的细胞系被冻结。 </li><li>从一个48小时的T - 25瓶（约1.5x10 6细胞/ mL）的旧文化的细胞在室温下5分钟1500转离心。丢弃上清后，细胞重新悬浮在2毫升冷冻结介质。两个1.0毫升细胞悬液卷从每个烧瓶放置在冷冻小瓶，立即冻结在-80 ° C，并转移到液态氮的翌日。 </li><li>从液氮保存的细胞恢复快速解冻，resuspending 10 mL新鲜培养液中的内容。另外，刚刚解冻的细胞在温暖的培养基上进行了一次洗涤之前删除二甲基亚砜10毫升的新鲜培养液resuspending。细胞培养随后通过前至少72 h。 </li></ol> 6。代表性的成果 自动取款机- / - T细胞线使用此协议，并指定DWJ - ATM - 1与抗CD3 - FITC，抗CD4 - APC和抗- CD8 - PE和流式细胞仪分析（图1）染色的特点。超过90％的T细胞，CD3，CD4和CD8 +三重阳性。 <img src="/files/ftp_upload/2598/2598fig1.jpg" alt="图1" /> 图1:使用该协议所开发的T -细胞系的表征。 / - -建立ATM的细胞表面标记的细胞株指定DWJ - ATM - 1的特点是染色与抗CD3 - FITC，抗CD4 - APC，抗- CD8 - PE和流式细胞仪分析。简单地说，1X10 6新鲜细胞清洗一次冷PBS（1500转/ 5分），孵育4 ° C为10分钟与200μL抗体鸡尾酒（每个抗体1:200稀释1％BSA的PBS。细胞可以立即进行检查，而新鲜或固定为以后的分析。固定细胞准备加入缓冲的福尔马林在中立在终浓度为4％（体积/体积），并在4 ° C孵育30分钟，离心收获，（1500转/ 5分钟），重悬于200μL1％BSA的PBS。共1 × 10 4细胞进行了分析使用BD流式细胞仪刀魂分析仪和CellQuest软件（BD公司，加利福尼亚州圣何塞） 。

<ol>
	<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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

12.2K 次观看.  Cornell University. 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。

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

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

科研

发育生物学

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

癌症研究

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

BLT人性化的鼠标使用一个基于干细胞的基因治疗肿瘤模型的建立

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

免疫与感染

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

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 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

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 ...

<meta charset="utf8"></head><body>使用野生型封闭 PCR 优化低频突变检测

Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting for drug resistance mutations and circulating tumor cell mutations during therapy. Traditional Sanger sequencing has 5%-20% wild-type mutation detection, so its limit of detection cannot meet the corresponding requirements. The wild-type blocking technologies that have been reported to overcome this include blocker displacement amplification (BDA), non-extendable locked nucleic acid (LNA), hot-spot-specific probes (HSSP), etc. These technologies use specific oligonucleotide sequences to block wild-type or recognize wild-type and then combine this with other methods to prevent wild-type amplification and amplify mutant amplification, leading to characteristics like high sensitivity, flexibility, and convenience. This protocol uses BDA, a wild-type blocking PCR combined with Sanger sequencing, to optimize the detection of RHOA G17V low-frequency somatic mutations, and the detection sensitivity can reach 0.5%, which can provide a basis for MRD monitoring of angioimmunoblastic T-cell lymphoma.

<meta charset="utf8"></head><body>作者聚焦：推进癌症组织中低频突变的检测

This article introduces the application of a low-frequency detection method based on Sanger sequencing in angioimmunoblastic lymphoma. Provide a basis for applying this method to other diseases.

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting ...

遗传学

Spontaneous Murine Model of Anaplastic Thyroid Cancer

Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy with a dismal prognosis. There is an urgent need for more in-depth research on the carcinogenesis and development of ATC, as well as therapeutic methods, since standard treatments are essentially depleted in ATC patients. However, low prevalence has hampered thorough clinical studies and the collection of tissue samples, so little progress has been achieved in creating effective treatments. We used genetic engineering to create a conditionally inducible ATC murine model (mATC) in a C57BL/6 background. The ATC murine model was genotyped by TPO-cre/ERT2; BrafCA/wt; Trp53ex2-10/ex2-10 and induced by intraperitoneal injection with tamoxifen. With the murine model, we investigated the tumor dynamics (tumor size ranged from 12.4 mm2 to 32.5 mm2 after 4 months of induction), survival (the median survival period was 130 days), and metastasis (lung metastases occurred in 91.6% of mice) curves and pathological features (characterized by Cd8, Foxp3, F4/80, Cd206, Ki67, and Caspase-3 immunohistochemical staining). The results indicated that spontaneous mATC possesses highly similar tumor dynamics and immunological microenvironment to human ATC tumors. In conclusion, with high similarity in pathophysiological features and unified genotypes, the mATC model resolved the shortage of clinical ATC tissue and sample heterogeneity to some extent. Therefore, it would facilitate the mechanism and translational studies of ATC and provide an approach to investigate the treatment potential of small molecular drugs and immunotherapy agents for ATC.

Here, we present a standard pipeline to obtain murine ATC tumors by spontaneous genetically engineered mouse models. Further, we present tumor dynamics and pathological information about the primary and metastasized lesions. This model will help researchers to understand tumorigenesis and facilitate drug discoveries.

间变性甲状腺癌的自发性鼠模型

Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy with a dismal prognosis. There is an urgent need for more in-depth research on the ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

隔离和不同的老年小鼠胸腺移植的移植。

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

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

摘要

探索更多视频

此视频中的章节

胸腺表征祖定居在小鼠胚胎使用

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

BLT人性化的鼠标使用一个基于干细胞的基因治疗肿瘤模型的建立

考试流式细胞仪检测胸腺阳性和阴性选择

对T细胞推导

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

间变性甲状腺癌的自发性鼠模型

隔离和不同的老年小鼠胸腺移植的移植。

胸腺淋巴瘤的T细胞系的推导 ATM - / - P53 - / -鼠标

摘要

探索更多视频

此视频中的章节

胸腺表征祖定居在小鼠胚胎使用

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

BLT人性化的鼠标使用一个基于干细胞的基因治疗肿瘤模型的建立

考试流式细胞仪检测胸腺阳性和阴性选择

对T细胞推导

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

间变性甲状腺癌的自发性鼠模型

隔离和不同的老年小鼠胸腺移植的移植。

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