Name: quantification of circulating pig-specific dna in the blood of a xenotransplantation model
Uploaded: 2020-09-22T14:00:00
Description: Watch this Scientific Journal Video about Quantification of Circulating Pig-Specific DNA in the Blood of a Xenotransplantation Model at JoVE.com

这项工作得到了中国国家重点研发项目（2017YFC1103704）、深圳市科技基金会（JCJ20170817172116272）、广东省高水平医院建设专项资金的资助。 （2019年），深圳市三明医药工程（SZSM201412020），深圳市高级医疗学科建设基金（2016031638），深圳市卫生计发委（SZXJ2017021）， SZXJ2018059）.

所有实验都是按照深圳市第二人民医院、深圳市第一附属医院机构审查委员会的有关指导意见和规定进行的。
1. 设计猪专用底剂
<ol><li>使用软件NCBI（www.ncbi.nlm.nih.gov）进行全基因组爆炸分析，以识别与人类、猴子或小鼠不同的www.ncbi.nlm.nih.gov。</li>
	<li>使用底像5的软件根据19个猪特异性基因（表1）设计底像。</li>
</ol>2. 隔离基因组DNA
<p class="jo...

<ol>
	<li>Buchman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+organ+failure.">Multiple organ failure.</a> Current Opinion In General Surgery. , 26-31 (1993).</li><li>Smith, M., Dominguez-Gil, B., Greer, D., Manara, A., Souter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+donation+after+circulatory+death:+current+status+and+future+potential.">Organ donation after circulatory death: current status and future potential.</a> Intensive care medicine. 45 (3), 310-321 (2019).</li><li>Lopez-Fraga, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+needed+Convention+against+trafficking+in+human+organs.">A needed Convention against trafficking in human organs.</a> Lancet. 383 (9936), 2187-2189 (2014).</li><li>Sykes, M., Sachs, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplanting+organs+from+pigs+to+humans.">Transplanting organs from pigs to humans.</a> Science immunology. 4 (41), 6298 (2019).</li><li>Reardon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+life+for+pig-to-human+transplants.">New life for pig-to-human transplants.</a> Nature. 527 (7577), 152-154 (2015).</li><li>Song, Z., Cooper, D. K., Mou, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+Regulation+Profile+of+Mature+MicroRNA+in+the+Pig:+Relevance+to+Xenotransplantation.">Expression and Regulation Profile of Mature MicroRNA in the Pig: Relevance to Xenotransplantation.</a> BioMed Research International. 2018, 2983908 (2018).</li><li>Chung, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4(+)+/CD8(+)+T-cell+ratio+correlates+with+the+graft+fate+in+pig-to-non-human+primate+islet+xenotransplantation.">CD4(+) /CD8(+) T-cell ratio correlates with the graft fate in pig-to-non-human primate islet xenotransplantation.</a> Xenotransplantation. 27 (2), 12562 (2020).</li><li>Yoon, C. H., Choi, S. H., Lee, H. J., Kang, H. J., Kim, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictive+biomarkers+for+graft+rejection+in+pig-to-non-human+primate+corneal+xenotransplantation.">Predictive biomarkers for graft rejection in pig-to-non-human primate corneal xenotransplantation.</a> Xenotransplantation. 26 (4), 12515 (2019).</li><li>Agbor-Enoh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+cell-free+DNA+as+a+biomarker+of+tissue+injury:+Assessment+in+a+cardiac+xenotransplantation+model.">Circulating cell-free DNA as a biomarker of tissue injury: Assessment in a cardiac xenotransplantation model.</a> The Journal of Heart and Lung Transplantation. 37 (8), 967-975 (2018).</li><li>Vito Dabbs, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+symptom+reports+useful+for+differentiating+between+acute+rejection+and+pulmonary+infection+after+lung+transplantation.">Are symptom reports useful for differentiating between acute rejection and pulmonary infection after lung transplantation.</a> Heart &amp; Lung. 33 (6), 372-380 (2004).</li><li>Miller, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+approaches+for+the+diagnosis+of+acute+cardiac+allograft+rejection.">Non-invasive approaches for the diagnosis of acute cardiac allograft rejection.</a> Heart. 99 (7), 445-453 (2013).</li><li>Mandel, P., Metais, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Les+acides+nucl&#233;iques+du+plasma+sanguin+chez+l'homme.">Les acides nucl&#233;iques du plasma sanguin chez l'homme.</a> Comptes Rendus des Seances de l Academie des Sciences. 142 (3-4), 241-243 (1948).</li><li>Okkonen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+cell-free+DNA+in+patients+needing+mechanical+ventilation.">Plasma cell-free DNA in patients needing mechanical ventilation.</a> Critical Care. 15 (4), 196 (2011).</li><li>Wagner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+DNA--new+potential+analyte+in+clinical+laboratory+diagnostics.">Free DNA--new potential analyte in clinical laboratory diagnostics.</a> Biochemia Medica. 22 (1), 24-38 (2012).</li><li>Schwarzenbach, H., Nishida, N., Calin, G. A., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+relevance+of+circulating+cell-free+microRNAs+in+cancer.">Clinical relevance of circulating cell-free microRNAs in cancer.</a> Nature Reviews Clinical Oncology. 11 (3), 145-156 (2014).</li><li>Mohiuddin, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+2C10R4+anti-CD40+antibody+therapy+is+critical+for+long-term+survival+of+GTKO.hCD46.hTBM+pig-to-primate+cardiac+xenograft.">Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft.</a> Nature Communications. 7, 11138 (2016).</li><li>Bettegowda, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+circulating+tumor+DNA+in+early-+and+late-stage+human+malignancies.">Detection of circulating tumor DNA in early- and late-stage human malignancies.</a> Science Translational Medicine. 6 (224), 24 (2014).</li><li>Dawson, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+circulating+tumor+DNA+to+monitor+metastatic+breast+cancer.">Analysis of circulating tumor DNA to monitor metastatic breast cancer.</a> New England Journal of Medicine. 368 (13), 1199-1209 (2013).</li><li>Newman, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultrasensitive+method+for+quantitating+circulating+tumor+DNA+with+broad+patient+coverage.">An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage.</a> Nature Medicine. 20 (5), 548-554 (2014).</li><li>Snyder, T. M., Khush, K. K., Valantine, H. A., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+noninvasive+detection+of+solid+organ+transplant+rejection.">Universal noninvasive detection of solid organ transplant rejection.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (15), 6229-6234 (2011).</li><li>De Vlaminck, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+monitoring+of+infection+and+rejection+after+lung+transplantation.">Noninvasive monitoring of infection and rejection after lung transplantation.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (43), 13336-13341 (2015).</li><li>Zhou, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+pig-specific+DNA+as+a+novel+biomarker+for+monitoring+xenograft+rejection.">Circulating pig-specific DNA as a novel biomarker for monitoring xenograft rejection.</a> Xenotransplantation. 26 (4), 12522 (2019).</li><li>Huo, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+mi+RNA+or+circulating+DNA+&#8212;Potential+biomarkers+for+organ+transplant+rejection.">Circulating mi RNA or circulating DNA &#8212;Potential biomarkers for organ transplant rejection.</a> Xenotransplantation. 26 (1), 12444 (2019).</li><li>Gadi, V. K., Nelson, J. L., Boespflug, N. D., Guthrie, K. A., Kuhr, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+donor+DNA+concentrations+in+recipient+serum+correlate+with+pancreas-kidney+rejection.">Soluble donor DNA concentrations in recipient serum correlate with pancreas-kidney rejection.</a> Clinical Chemistry. 52 (3), 379-382 (2006).</li></ol>

量化猪循环DNA是监测异种移植的免疫排斥的可行方法。Gadi等人24 日发现，急性排斥患者血液中的供体衍生循环DNA（ddcfDNA）含量明显高于无排斥患者的血液中。这些研究表明，ddcfDNA可能是监测器官移植损伤的常见生物标志物。近年来，qPCR由于操作简单、自动化程度高、灵敏度高、特异性好、成本低，在核酸分析中应用日益深入。
本研究中，根据生物信息学?...

器官衰竭是导致1人死亡的主要原因之一。细胞、组织和器官移植是治疗器官衰竭的有效方法。然而，捐赠器官的短缺限制了这种方法的临床应用3，4。研究表明，猪可以作为人体器官的潜在来源进行临床移植5，6。然而，跨物种器官移植面临危险的免疫排斥。因此，监测异种移植的免疫排斥至关重要。目前，免疫排斥的临床监测主要取决于患者的体征和症状，以及实验室测试（如活检、免疫生化分析和超声波）7、8、9。但是，这些监视方法有许多缺点。患者免疫排斥的体征和症状通常晚10晚出现，不利于早期发现和早期干预;活检有侵入性11的缺点，患者不容易接受;免疫生物化学分析缺乏敏感性或特异性，超声波是辅助的，而且价格昂贵。因此，寻找一种有效、方便的方法来监测免疫排斥是当务之急。
循环DNA是一种在血液中发现的细胞外DNA。曼德尔和梅泰斯12号在1948年首次报告了周围血液中存在循环DNA。在正常的生理条件下，健康人血液中的循环DNA在基线上相对较低。然而，在一些病理，如肿瘤，心肌梗死，自身免疫性疾病，移植排斥，循环DNA水平在血液中可以显著增加13，14由于大量释放循环DNA引起的凋亡和坏死因子。循环DNA的起源与凋亡和坏死15有关，这是异种移植排斥16的特征。
循环DNA已被证明是一种微创生物标志物，用于检测癌症17，18，19。供体衍生循环DNA的高直通测序是检测器官移植20、21后排斥的可靠方法。然而，这种方法要求高浓度和中提取的DNA质量。除了高昂的成本和时间消耗外，DNA要求使这种方法没有资格用于常规临床应用。供体衍生的循环DNA可以通过定量实时PCR（qPCR）精确量化，它既具体又敏感。因此，通过qPCR量化猪循环DNA是监测异种移植免疫排斥的可行方法。这具有较少的侵入性、高度敏感和特异性、低成本和节省时间。猪和人类在基因上是分开的，基因组序列完全不同（图1）。因此，循环的猪DNA可以释放到接受者的血液后异种移植由于异种排斥。CpsDNA 可以通过 qPCR 精确量化，在接受者血液中使用物种特异性底剂。此前，我们已经论证了cpsDNA作为异种移植22，23的生物标志物的理由和可行性。在这里，我们披露更多的实验技巧和细节。实验由以下步骤组成。首先，设计了猪特异性底剂，分离了基因组DNA，用于通过常规PCR验证底源剂的特异性。其次，构建cpsDNA的标准曲线，将cpsDNA与样本血隔离。最后，使用qPCR对循环猪特异性DNA进行量化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td ><a>Agarose</a></td>
			<td >Biowest, Barcelona, Spain</td>
			<td >111860</td>
			<td ></td>
		</tr>
		<tr >
			<td >BamHI-HF</td>
			<td >New England Biolabs, Ipswich, Mass, USA</td>
			<td >R3136S</td>
			<td ></td>
		</tr>
		<tr >
			<td >1.5 mL microcentrifuge tube</td>
			<td >Axygen&#160;Biosciences, Union City, CA, USA</td>
			<td >MCT-150-C</td>
			<td ></td>
		</tr>
		<tr >
			<td >0.2 mL PCR tube</td>
			<td >Axygen&#160;Biosciences, Union City, CA, USA</td>
			<td >PCR-02-C</td>
			<td ></td>
		</tr>
		<tr >
			<td >C57BL/6 Mice</td>
			<td >Medical Animal Center of Guangdong Province,&#160; China</td>
			<td ></td>
			<td >8~10 weeks</td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Thermo&#160;Fisher&#160;Scientific, Walt- ham, MA, USA</td>
			<td >Micro 21R</td>
			<td ></td>
		</tr>
		<tr >
			<td >2-Log DNA Ladder</td>
			<td >New England Biolabs, Ipswich, Mass, USA</td>
			<td >N3200S</td>
			<td >0.1&#8211;10.0 kb</td>
		</tr>
		<tr >
			<td >Marker I&#160;</td>
			<td >Tiangen, Beijing, China</td>
			<td >MD101-02</td>
			<td >0.1&#8211;0.6 kb</td>
		</tr>
		<tr >
			<td >DNA Mini Column(HiBind DNA Mini Columns)</td>
			<td >Omega Bio-tek, Norcross, GA, USA</td>
			<td >DNACOL-01</td>
			<td ></td>
		</tr>
		<tr >
			<td >DNA loading buffer</td>
			<td >Solarbio, Beijing, China</td>
			<td >D1010</td>
			<td ></td>
		</tr>
		<tr >
			<td rowspan="2" >E.Z.N.A.Plasmid DNA Mini Kit I and E.Z.N.A. Plasmid DNA Mini Kit II</td>
			<td rowspan="2" >Omega Bio-tek, Norcross, GA, USA</td>
			<td >D6942</td>
			<td rowspan="2" ></td>
		</tr>
		<tr >
			<td >D6943</td>
		</tr>
		<tr >
			<td >EcoR I</td>
			<td >Takara&#160;Bio, Shiga, Japan</td>
			<td >&#160;1040S</td>
			<td ></td>
		</tr>
		<tr >
			<td >Female Bama mini pigs</td>
			<td >BGI Ark Biotechnology, Shenzhen, China</td>
			<td ></td>
			<td >2~4 months</td>
		</tr>
		<tr >
			<td >Genomic DNA Extraction Kit &#8544;</td>
			<td >Tiangen, Beijing, China</td>
			<td >DP304-02</td>
			<td ></td>
		</tr>
		<tr >
			<td >SYBR Green Realtime PCR Master Mix</td>
			<td >Toyobo, Osaka, Japan</td>
			<td >QPK-201</td>
			<td ></td>
		</tr>
		<tr >
			<td >Gel Doc XR</td>
			<td >Bio-Rad, Hercules, USA</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Male cynomolgus monkeys</td>
			<td >Guangdong Landau Biotechnology, Guangzhou, China</td>
			<td ></td>
			<td >8 years</td>
		</tr>
		<tr >
			<td >Nucleic acid dye(Gelred)</td>
			<td >Biotium, Fremont, USA</td>
			<td >42003</td>
			<td ></td>
		</tr>
		<tr >
			<td >polymerase(Premix Taq)</td>
			<td >Takara&#160;Bio, Shiga, Japan</td>
			<td >RR901A</td>
			<td ></td>
		</tr>
		<tr >
			<td >pMD19-T plasmid</td>
			<td >Takara&#160;Bio, Shiga, Japan</td>
			<td >D102A</td>
			<td ></td>
		</tr>
		<tr >
			<td >qPCR machine</td>
			<td >Applied Biosystems QSDx, Waltham, USA</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Serum/Circulating DNA Extraction Kit</td>
			<td >Tiangen, Beijing, China</td>
			<td >DP339</td>
			<td ></td>
		</tr>
		<tr >
			<td >TAE</td>
			<td >sangon Biotech, Shanghai, China</td>
			<td >B548101</td>
			<td ></td>
		</tr>
	</tbody>
</table>

quantification of circulating pig-specific dna in the blood of a xenotransplantation model

异种移植是治疗器官衰竭的可行方法。然而，如何有效地监测异种移植的免疫排斥是医生和研究人员面临的一个问题。本手稿介绍了一种简单有效的方法来监测猪对老鼠细胞移植模型和猪对猴子动脉贴片移植模型中的免疫排斥。循环DNA是一种潜在的非侵入性生物标志物，用于器官损伤。在这项研究中，通过定量实时PCR（qPCR）监测猪特异性DNA（cpsDNA） 在异种移植排斥过程中。在该协议中，设计了猪特异性底剂，构建了含质粒的猪特异性DNA片段，并建立了定量标准曲线。然后，在猪对小鼠细胞移植模型和猪对猴子动脉贴片移植模型中，使用物种特异性底剂通过 qPCR 对 cpsDNA 进行量化。该方法的价值表明，它可以作为一种简单、方便、低成本、侵入性较小的方法来监测异种移植的免疫排斥。

在该协议中，设计了猪特异性底剂，构建了含有质粒的猪特异性DNA片段，并建立了定量标准曲线（图4）。19种底数的物种特异性得到PCR的确认。然后，在猪对小鼠细胞移植模型和猪对猴子动脉贴片移植模型中，使用物种特异性底像（底像#4和底像#11）通过qPCR对cpDNA进行量化。
Agarose电泳用于从上述样品中分离放大的DNA片段，然后在紫外线成像器中可视化?...

Watch this Scientific Journal Video about Quantification of Circulating Pig-Specific DNA in the Blood of a Xenotransplantation Model at JoVE.com

Quantification of Circulating Pig-Specific DNA in the Blood of a Xenotransplantation Model

在该协议中，设计了猪特异性底剂，构建了含质粒的猪特异性DNA片段，并建立了定量标准曲线。使用物种特异性底法，cpsDNA在猪对小鼠细胞移植模型和猪对猴子动脉贴片移植模型中通过qPCR进行量化。

异种移植模型中循环猪特异性DNA的定量

Xenotransplantation is a feasible method to treat organ failure. However, how to effectively monitor the immune rejection of xenotransplantation is a ...

异种移植是治疗的可行方法，然而，有效监测异种移植的免疫排斥是医生和研究人员面临的一个问题。该协议是一种简单、方便、成本低、侵入性较小的方法来监测异种转化的免疫排斥。该技术可用于异种移植领域，包括猪对人岛移植、肾移植、肝移植和心脏移植。首先将500微升血液样本转移到不同的微离心管中。在管中加入20微升蛋白酶K和500微升的解液缓冲液。然后彻底摇动它们混合。将管子在56摄氏度的水浴中放入10分钟，在孵育过程中摇动两到三次，直到溶液变得清晰，将它们短暂离心，从管盖内壁上取出液体珠子。然后加入500微升无水、乙基醇，彻底摇动。将混合物转移到吸附列中。然后将柱子离心两分钟，丢弃废液。在每个吸附柱中加入800微升冲洗溶液，并离心机一分钟。将柱在室温下保持几分钟，以干燥剩余的冲洗溶液。将吸收柱转移到干净的离心管中。在吸附膜中间加入50微升洗脱缓冲液，将其放在室温下两至五分钟，然后离心1分钟。根据手稿方向准备 PCR 主混合，然后将混合的 23 微升添加到每个 0.6 毫升微离心管中。在每个管中加入两个微升的基因组DNA，并小心地卡博特。然后短暂混合和离心。将管子放在 PCR 循环器中，然后开始放大。当反应完成后，执行阿加糖电泳。将1.2克阿加糖加入含有100毫米TA的烧瓶中，在微波炉中煮5分钟。一旦阿加罗斯冷却到约70摄氏度，将5微升的核酸染料加入烧瓶中，慢慢倒入盘子中，在室温下保持，直到凝固成凝胶。在每个样品中加入5微升和2-LogDNA梯子或DNA标记1，加入1.2亿个码头的阿加罗斯凝胶和电泳井，直到带子分离。使用紫外线成像器可视化凝胶。执行文本手稿中描述的转换后，使用 EcoR I 和 Bam HI 通过双限制性酶消化验证质粒。将消化的产品与1%的阿加糖电泳分离，将凝胶暴露在紫外线下。使用双蒸馏水，用猪DNA片段稀释浓缩质粒，每毫升2倍10至11份。要建立标准曲线，请准备一个 qPCR 主混合，如文本手稿中所述，并将混合的 40 微升添加到八管条的每个管中。加入10微升不同浓度的标准DNA，盖住管子，然后短暂混合和离心。将管子放在 qPCR 机器中并执行放大。使用 EDTA 管从猪到猴子动脉贴片模型收集大约 400 微升的血液样本，或从猪到老鼠细胞移植模型中收集大约 100 微升的血液样本。然后将样品转移到1.5毫升离心管。在低温和高速下离心，从血液样本中去除血细胞。将上经剂转移到新的1.5毫升离心管中，并在16，000次G下离心，在10分钟内清除细胞碎片。将上流液转移到新的1.5毫升离心管中，在循环DNA提取试剂盒时，使用商业血清提取循环DNA。将循环DNA的体积压缩到40微升，并将其储存在负20摄氏度。执行 qPCR 以量化循环猪特异性 DNA。猪特异性底像被设计并用于通过qPCR，在猪对老鼠细胞移植模型和猪到猴子动脉补丁，移植模型中量化循环猪特异性DNA。阿加罗斯电泳用于分离放大的DNA片段。使用PCR，鉴定了放大猪基因组DNA的底剂特异性。其次，这些遗传基因在猴子或人类基因组DNA队列或小鼠基因组DNA队列中的物种特异性得到确认。最后两个物种特异性底法，用于放大猪DNA在猪到猴子动脉补丁和猪到小鼠，细胞移植模型。在尝试此协议时，要记住的最重要的事情是，应防止各种污染。这项技术为研究人员探索异种移植中的新方法铺平了道路，因为它是一种简单的低成本和非侵入性方法来监测异种移植的免疫排斥。

Research

JoVE Journal

Biology

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

微流控自动化血液循环核细胞富集的裂解协议

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and ...

科研

生物学

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

人类干细胞异种移植到鸡胚

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

全安装胚胎肢体皮肤血管的免疫组织化学分析：一个模型系统，研究在胚胎血管分支形态发生

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

使用可视化的DNA复制DNA纤维的技术，在脊椎动物的模型系统DT40

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Sampling Blood from the Lateral Tail Vein of the Rat

从大鼠的侧尾静脉血液取样

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

药物与DNA相互作用的细胞全基因组映射与COSMIC（小分子交联，染色质隔离）

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...