Name: using quantitative real-time pcr to determine donor cell engraftment in a competitive murine bone marrow transplantation model
Uploaded: 2013-03-07T13:00:00
Description: Watch this Scientific Journal Video about Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model at JoVE.com

我们感谢查尔斯·格林伯格博士利用他的实时PCR仪。 MUSC霍林斯癌症中心的启动资金，霍林斯癌症中心的ACS IRG，ASCO征服癌症基金会事业发展奖，美国国立卫生研究院1K08HL 103780-01A1，和美国国立卫生研究院3P30CA138313-01S3这项工作是支持的。的内容完全是作者的责任，并不一定代表官方意见的国家机构的健康或其他资金剂。

1。骨髓细胞分离<ol><li>安乐死男性捐赠者FVB / NJ小鼠和雌性FVB / NJ小鼠使用CO 2的方法，随后用颈脱位。竞争力的细胞将被用作阴FVB / NJ骨髓细胞。 </li><li>用小剪刀和镊子，解剖小鼠和股骨和tibiaes，将它们放置在60毫米组织培养皿含6毫升冰冷的5％热灭活FBS培养液。使用kimwipe组织，去除肌肉和其他组织。在盘子里，切断两端的每个骨干。 </li><li>用23G针3毫升注射器的一端连接的骨，冲洗掉?...

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我们目前的研究目标是提供观众一个基于PCR技术定量供体细胞移植在竞争激烈的小鼠骨髓移植模型。一些研究已经用RT-PCR检测供体细胞移植模型9-10。第一博士Schwarzenberger的研究小组开发出了小鼠骨髓移植模型，采用实时PCR扩增Y染色体特定的8。这种方法被用来研究GLI-1在造血干细胞的功能活动11。然后，我们进一步修改了协议，在可视化的格式，并提供了一​​个详细的，...

小鼠骨髓（BM）移植模型最早是在20世纪60年代1。这个模型已被广泛用于造血干细胞（HSC）在宿主受体小鼠的生物学研究。小鼠骨髓移植模型为我们提供了宝贵的知识HSC功能及其调控，造血干细胞的研究是必不可少的。异基因骨髓移植模型，例如C57BL/6J H2B-BALB / C的H2D或同源移植模型如C56Bl/6J CD45.2 B6.SJL CD45.1中使用的许多实验室研究基因的功能，影响HSC活动2 HSC功能3或移植相关的疾病，如移植物抗宿主病（GvHD）4上的药物治疗。 通常用于区分来自供体的细胞，从收件人-起源的细胞的细胞表面标志物，如MHC单倍型或CD45.1。 C57BL/6J H2B，CD45.2 </suP&gt;，BALB / C H2D和B6.SJL的CD45.1是最常用的小鼠品系，在骨髓移植的供体细胞的贡献，因为可以很容易地用流式细胞仪，CD45.1 与 CD45.2或H2B与评估H2D。 FVB / NJ 5 C3H然而，许多其他菌株也经常被用来生成基因工程转基因或基因敲除小鼠。这些小鼠可回交自交系和保持在混合的遗传/ MHC背景。在这种情况下，确定供体细胞移植和造血干细胞的功能可能是困难的，因为供体特异性的细胞表面标志物可能无法使用。 Y-染色体特异DNA探针来检测Southern杂交性别不匹配的骨髓移植供体雄性细胞最早是由美和博士的研究小组6。然后，在实时PCR的性别决定区Y被认为是准确的和高度特异性的方法来定量毫安乐胎儿细胞在母体血液系统7。这个概念被改编由博士Schwarzenberger的研究小组开发的实时PCR技术在小鼠骨髓移植模型，以确定供体细胞植入8。我们进一步修改FVB / NJ小鼠骨髓移植模型中的体细胞移植的测量方法。这种方法是目前广泛使用于本集团PIM1激酶在造血干细胞生物学作用的研究。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> RPMI 1640 </td><td> Hyclone公司</td><td> SH30255.01 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen公司</td><td> 16140-017 </td><td>热灭活</td></tr><tr><td>氯化铵</td><td> MP Biomedicaals </td><td> 194806 </td><td></td></tr><tr><td>碳酸氢钾</td><td>费舍尔</td><td> P184-500 </td><td></td></tr><tr><td>的QIAamp DNA血液MINIKIT </td><td> Qiagen公司</td><td> 51106 </td><td></td></tr><tr><td>细胞过滤器</td><td> BD生物科学</td><td></td><td></td></tr><tr><td> iQ SYBR Green超</td><td> Biorad公司</td><td> 170-8882 </td><td></td></tr><tr><td> iQ5实时PCR仪</td><td> Biorad公司</td><td></td><td></td></tr><tr><td>光谱光度计</td><td>的NanoDrop ND-1000 </td><td> ND-1000 </td><td></td></tr></tbody></table>

using quantitative real-time pcr to determine donor cell engraftment in a competitive murine bone marrow transplantation model

小鼠骨髓移植模型提供了一个重要的工具，在测量造血干细胞（HSC）功能，并确定基因/分子，调节造血干细胞。在这些移植模型系统，是由这些细胞的能力，嫁接和重组致死剂量照射的受体小鼠的造血干细胞的功能。一般情况下，供体细胞的贡献/植活测定用流式细胞仪供体特异性的细胞表面蛋白的抗体。然而，该方法在很大程度上依赖于特异性和区分来自供体的细胞的细胞表面标志物的能力，从收件人起源的细胞，这可能不是可用于所有的小鼠品系。考虑到各种不同的背景在市场上的转基因小鼠品系，该细胞表面/流量流式细胞仪为基础的方法具有显着的局限性，特别是在缺少界定良好的从同类系收件人策单独的供体细胞的表面标志物的小鼠品系LLS。在这里，我们报道了基于PCR的技术来确定移植的受体小鼠的体细胞移植/贡献。男性捐赠者的骨髓造血干细胞移植到致死剂量照射的同源雌性小鼠。在不同时间点移植后外周血标本收集。在实验结束后获得的骨髓标本。分离基因组DNA和Y染色体的特定基因，Zfy1，采用实时定量PCR扩增。在女性受体小鼠的植入男性捐赠者来源的细胞对标准曲线的男性与女性的DNA与已知的百分比计算。 Bcl2蛋白被用作参照基因正常化的总DNA量。我们的数据表明，这种方法可靠地确定供体细胞植入，并提供了一​​个有用的，但简单的方法，测量造血干细胞重建小鼠骨髓移植模型。我们的方法可以在大多数实验室常规进行的，因为没有需要昂贵的设备，如流式细胞仪。

图1和图2显示对男性的DNA的百分比，平均2-ΔCT值绘制标准曲线的例子， 图1A中显示的特定的熔点温度Bcl2和Zfy1的扩增子定位于78.5℃和88.5℃，分别。 Bcl2蛋白被用作参照基因正常化加载的每个PCR反应的DNA的总量。 Bcl2的扩增曲线（日志视图），对于每个标准样品合并男性DNA浓度与彼此独立，表示加载的DNA（ 图1B）的等量。然而，Zfy1迁移的扩增曲线的左?...

Watch this Scientific Journal Video about Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model at JoVE.com

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

确定供体细胞移植在小鼠骨髓移植模型中，缺乏定义的表型标记提出了挑战。我们描述了一种方法来量化男性捐赠者的细胞植入女性移植受体小鼠。此方法可用于研究HSC功能在所有的小鼠品系。

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Research

JoVE Journal

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A Mouse Model of in Utero Transplantation

A Mouse Model of in Utero Transplantation

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

科研

医学

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

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小鼠角膜移植模型，研究实体器官移植的最常见形式

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Ex Situ Normothermic Machine Perfusion of Donor Livers

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供肝易地常温灌注机

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

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Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...