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 ...

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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

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

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)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

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

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

酪氨酸激酶抑制剂的临床前评价急性白血病的治疗

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

用改进的袖套法小鼠颈部心脏移植模型

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

脑脊液的microRNA分析采用实时定量PCR技术

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

小鼠角膜移植模型，研究实体器官移植的最常见形式

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time 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 death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

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 ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

使用改性非缝合铐技术的宫颈主动脉移植模型

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.