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，冲洗掉骨髓与5％热灭活FBS的RPMI1640入菜。个体骨髓组织中重复使用同一个针头的愿望。细胞悬浮液转移到15毫升离心管中。 </li><li>降速的细胞在400×g离心5分钟，除去上清液，细胞悬浮在1ml室温红血细胞溶解缓冲液（155毫米Potassium碳酸氢钠，氯化铵的10mM，0.1mM的EDTA，PH = 7.4），并在室温下孵育5分钟，然后加入5-10毫升，用5％热灭活的FBS的RPMI 1640。 </li><li>通过细胞通过细胞过滤网。收集流过到新的试管中。自旋向下的5分钟，于400×g。去除上清，细胞沉淀，不应该包含任何红色。红色的情况下指示一个完整的红血细胞的去除。与5％热灭活的FBS，细胞沉淀重悬在10毫升的RPMI1640。轻轻涡旋以确保细胞悬浮液是完全均匀的。分装在一个血球细胞计数。计算多少体积所需的骨髓移植和等分试样足够的细胞的细胞，并与竞争对手的雌性细胞在以5:2的比例混合男性供体细胞。降速在400×g离心5分钟，用PBS洗涤，重悬浮在PBS中的供体细胞的最终浓度在5×10 6个 / ml和竞争对手的细胞以2×10 6个 </su对&gt; /毫升。 </li></ol> 2。竞争骨髓移植<ol><li>照射雌性受体小鼠（8-12周龄），铯-137伽玛射线散热器在一个单一的剂量为11Gy骨髓移植前的4-6小时。 </li><li>放置在小鼠阻挡照射的雌性小鼠。经尾静脉注入混合的供体和竞争对手的细胞在0.1毫升的总体积，使得每个鼠标接收5×10 5个供体细胞和2×10 5的竞争对手骨髓细胞。 </li></ol> 3。样品采集情侣周后，骨髓移植，收集雌性受体小鼠外周血样本（约50微升）从麻醉状态下的眼窝出血。样品被收集到EDTA涂层管。 BM样品也被收集在实验结束时，通常至少为4个月后移植，用相同的方法与前面描述的（步骤1），通常为20-40％的1胫骨骨髓细胞足以让足量的DNA。也收集血液或骨髓样本从年龄匹配的正常雌性和雄性小鼠制备标准曲线。 4。基因组DNA提取<ol><li>加入4倍体积的:室温红细胞裂解缓冲液（200微升），各血液试样。搅拌均匀，室温孵育5分钟。加入1毫升的PBS，然后停止旋转，以去除大部分的裂解RBC的。 </li><li>使用血液DNA提取试剂盒（QIAmp DNA血液提取试剂盒）提取DNA。预温（AE）的洗脱缓冲液在37℃下洗脱的DNA，以提高产率。同样是孤立的男性和女性DNA的标准曲线。 </li><li> （可选）的基因组DNA可以进一步纯化，或在3 M醋酸钠（pH = 5.5）的存在下，用乙醇沉淀法浓缩。然后将DNA沉淀再悬浮在40-50微升蒸馏水立即进行分析，或储存在-20℃下，以备将来使用。 </li><li>测量的DNA concentr"ATION的NanoDrop ND-1000光谱光度计。样品OD 260/280之间的,1.8-2 .0用于进一步的分析。 </li><li>稀释的DNA与DNA酶游离H 2 O以4ng/μl在总体积为50μl。 </li></ol> 5。标准曲线的制备男性DNA和女性DNA稀释4纳克/微升的浓度，并根据表1的DNA混合物中<table border="1"><tbody><tr><td> ％的女性背景的男性DNA </td><td> 4ng/μl男性DNA的体积（μl） </td><td> 4ng/μl女性DNA的体积（μl） </td><td> 总体积（μl） </td></tr><tr><td> 0.2 </td><td> 1 </td><td> 499 </td><td> 500 </td></tr><tr><td> 0.5 </td><td> 1 </td><td> 199 </td><td> 200 </td></tr><tr><td> 2.5 </td><td> 5 </td><td> 195 </td><td> 200 </td></tr><tr><td> 12.5 </td><td> 25 </td><td> 175 </td><td> 200 </td></tr><tr><td> 50 </td><td> 100 </td><td> 100 </td><td> 200 </td></tr><tr><td> 87.5 </td><td> 175 </td><td> 25 </td><td> 200 </td></tr><tr><td> 100 </td><td> 200 </td><td> 0 </td><td> 200 </td></tr></tbody></table> 表1。样品制备标准曲线。正常男性和女性的FVB / NJ小鼠在年龄8 12wks的牺牲。收集血液和骨髓细胞。从雄性细胞和雌性细胞的DNA中分离出，并将其再悬浮在浓度4ng/μl。男性和女性的DNA在不同的比例混合，以产生DNA标准样品混合物。 6。实时PCR <ol><li>设置PCR SYBR Green超与引物和基因组DNA的试剂混合的反应板。反应体积（20微升）包含400 nM的各引物和5微升的血细胞的基因组DNA（4ng/μlX5的微升= 20毫微克）在每一个反应。 DNA桑普成立于一式三份文件和标准。引物序列示于表2。 </li></ol><table border="1"><tbody><tr><td> 基因名称 </td><td> 前 </td><td> 逆转 </td></tr><tr><td> Bcl2的</td><td> 5&#39;-AAGCTGTCACAGAGGGGCTA </td><td> 5&#39;-CAGGCTGGAAGGAGAAGATG </td></tr><tr><td> Zfy1 </td><td> 5-TGGAGAGCCACAAGCTAACCA的</td><td> 5&#39;-CCCAGCATGAGAAAGATTCTTC </td></tr></tbody></table> 表2中。RT-PCR引物序列Bcl2和Zfy1小鼠。 <ol start="2"><li>进行PCR反应，使用Biorad公司iQ5 PCR机具备以下条件:95℃3分钟，42个扩增循环:95°C为10秒，58℃下为25秒和72℃，持续15秒，然后通过熔融曲线步骤。 </li><li>获取由Bio-Rad iQ5 2.1标准版光学系统的循环阈值（Ct）值。计算ΔCT（CT Zfy1 &lt;/ SUP&gt;-CT Bcl2的 ）值，和Zfy1表达水平的计算公式为2-δCT值的。 </li><li>建立标准曲线，从已知的男/女标准混合使用2-ΔCT阅读的每个系列反应。所产生的标准曲线绘制2-ΔCT值的平均值的一式三份的已知％男性DNA的混合物用线性回归拟合。 </li></ol>

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我们没有竞争披露的财务权益。

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，一步一步的实验方案。我们的协议的可视化演示，使观众能够轻松地按照我们的协议。我们还比较了从骨髓样本与DNA从外周血基因组DNA的PCR结果。供体细胞植入从骨髓和血液样本的数据是基本一致的，但是，通常较小的变化被发现在骨髓样本。这是可能是由于在外周血中，可能会损害的血液样本中的基因组DNA的纯度/质量红细胞裂解后残留血红蛋白。 与我们的PCR为基础的技术有几个优点量化供体细胞的植入。第一;我们的技术可以在大多数的实验室中进行。有没有必要专门的和昂贵的设备以外的实时PCR机。我们进一步修改PCR反应成分和使用，而不是荧光定量SYBR Green超。这种变化进一步降低了成本和费用，我们消除了荧光标记的探针。第二，我们的技术是可靠，重现性好。我们使用Bcl2的参照基因正常化反对DNA定量试剂盒的总DNA量。 Bcl2的基因，因为:1）没有位于Y染色体上，这种基因是使用。因此，其表达不影响男性捐赠者的细胞植入。 2）。根据目前的移植条件Bcl2的不规范。为SH自己的在图1中，Bcl2的基因产生一个可靠的和高度一致的控制独立加载的男性DNA量的总DNA加载。 zfy1睾丸决定区域12内是一个锌指蛋白基因，并位于Y染色体上的。据报，该基因的表达水平的Zfy1很好地对应于男性细胞在小鼠移植模型8的 DNA的量。第三，我们的PCR为基础的技术是高度敏感的，而且可以检测到供体细胞移植在&lt;1％。此外，样品可以保存以供日​​后分析的很长一段时间。与此相反，传统的基于流式细胞术的方法需要立即样本处理。最后，我们的方法是独立于小鼠品系，并可以用于遗传背景，缺乏明确定义的分开从受体细胞的供体细胞的表面标志物。这是这种方法的最重要的优势。 根据我们目前的实验条件下（供体/竞赛或比率是5/2），我们周围观察到80-90％的供体的植入在16周使用基于PCR的方法。我们的数据是相当与传统，流式细胞仪为基础的方法，表明〜95％的供体细胞植入16周后移植供体（CD45.2），/竞争对手（CD45.1）的比例为4:1 2。与常规的细胞表面标志物的方法，该方法使用检测供体细胞在受体小鼠相比，我们的方法的主要缺点是无法分析在各种血细胞，如B细胞，T细胞或粒细胞亚群的植入。然而，如果需要的话，它是能够收集每个人口的细胞，用细胞分选技术，然后受用于RT-PCR的样品，以确定每个细胞种群中的供体细胞的贡献。由于供体细胞植入计算出标准曲线的基础上，的样品读出标准曲线的斜率和y轴截距是完全依赖于。这是可能的，我们可以得到范围外READOUT（ 即超过100％的植入）。然而，这些"假阳性或假阴性"结果可以可以避免的，如果:1）测试样品和标准曲线样品的DNA分离程序是相同的，以及2）标准曲线的选择的范围是接近预期的样本读数。此外，因为可能会出现在每个PCR反应中的差异和在PCR仪的校准状态，标准曲线样品应当被包括在每个PCR板，以避免每个PCR反应之间的差异。 最后，我们使用我们的PIM三KO小鼠证实了我们的技术。与以前的报告13，我们发现，从皮姆三基因敲除小鼠的供体造血干细胞是有缺陷的重组致死剂量照射的受体小鼠。

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侧，与男性的DNA样品中的量的增加（ 图1C）。为了验证我们的方法，我们测试的男性捐赠者的细胞植入WT 与 PIM三基因敲除小鼠（TKO）在不同时间点移植后小鼠骨髓细胞移植。周边的血（6wks）和BM的样品（16wks）瓦特<html> ERE进行了分析。造血干细胞从PIM将军澳小鼠有缺陷的重组致死剂量照射的小鼠（AN，N 等人 ，稿件修改）。正如预期的那样，与皮姆将军澳细胞移植的受体小鼠移植小鼠与野生型细胞（ 图3）相比，有低得多的百分比的雄性细胞。 <img alt="图1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50193/50193fig1highres.jpg" src="/files/ftp_upload/50193/50193fig1.jpg" /> 图1（A）代表从多个样品的熔解曲线与不同的男性DNA百分比。在78.5的峰表明了Bcl2的扩增，而在88.5℃的峰值C表示扩增ZFY-1。代表多个样品的扩增曲线，与不同的男性DNA Bcl2的（B）和Zfy1（C），CT:循环阈值的百分比。="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50193/50193fig1large.jpg"目标="_blank"&gt;点击这里查看大图。 <img alt="图2" src="/files/ftp_upload/50193/50193fig2.jpg" /> 图2。男性DNA百分比的RT-PCR检测的标准曲线。男性和女性DNA的混合物从骨髓细胞获得的样品（20 ng的DNA在20μl的总体积）中描述的协议用于RT-PCR进行了处理。 ΔCT（衣原体Zfy1-CT Bcl2蛋白 ）为个别样品的平均值进行了计算和2-δ的CT（Y-轴）对男性DNA百分比（X-轴）作图（范围从0.2％到100％），用线性回归恰当不过了。 R2代表的决定系数。 <img alt="图3" src="/files/ftp_upload/50193/50193fig3.jpg" /> 图3。竞争骨髓移植经验riments。 5×10 5总骨髓细胞从男性的WT或年龄匹配的男性的，皮姆三KO（TKO）随着2×10 5女选手骨髓细胞的小鼠被移植到照射的女主持人。外周血（PB）嵌合体在第​​6周和BM嵌合体在移植后16周，估计由男性的DNA的百分比通过RT-PCR为谴责在协议（* P &lt;0.05，** P &lt;0.01）。

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 ...

using-quantitative-real-time-pcr-to-determine-donor-cell-engraftment

Nanyang Technological University

Research

JoVE Journal

Medicine

15.3K 次观看.  Medical University of South Carolina. 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。

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

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

骨髓增殖性疾病的转导移植小鼠模型

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

科研

癌症研究

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

结合活体荧光显微镜 (IVFM) 和遗传模型研究造血细胞对骨髓生态位的植入动力学

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

发育生物学

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

竞争性移植评价造血干细胞健身

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

免疫与感染

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

多光谱定量分析继荧光组织移植的细胞起源，类型和相互作用的可视化

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

医学

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

单细胞指数分型与内皮细胞小生境联合培养对胚胎造血干细胞前体的克隆分析

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

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

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic 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 ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

CXCR4在Murine B-1a细胞上的逆转录病毒过度表达和靶向B-1a细胞向骨髓和IgM生产转移的采用转移

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

小鼠骨髓移植程序研究克隆造血症

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

<meta charset="utf8"></head><body>股骨内注射用于活小鼠骨髓研究

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

<meta charset="utf8"></head><body>使用活小鼠进行简化的股骨内注射允许连续骨髓分析

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

使用活小鼠进行简化的股骨内注射允许连续骨髓分析

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...