Name: measurement of bk-polyomavirus non-coding control region driven transcriptional activity via flow cytometry
Uploaded: 2019-07-13T14:00:00
Description: Watch this Scientific Journal Video about Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry at JoVE.com

作者感谢芭芭拉·布莱克曼的出色技术援助。这些研究得到了杜伊斯堡大学埃森医学院的IFORES项目以及鲁尔大学和默卡托研究中心(MERCUR)的RIMUR项目的支持。作者感谢约尔根-曼乔特-斯蒂夫通博士奖学金的海伦·塞尔兹尼格和不断的支持。患者材料的收集和使用已获得杜伊斯堡-埃森大学医学院伦理委员会的批准(14-6028-BO)。

本议定书遵循杜伊斯堡-埃森大学医学院伦理委员会(14-6028-BO)批准的人类研究准则。
1. 采集血液或尿液样本,分离多瘤病毒DNA
<ol><li>在EDTA管中采集至少3 mL的血液,或在采集管中收集尿液。</li>
	<li>将样品在2,500 x g下离心15分钟。如果需要,将等离子移液器放入新管中,将等离子样品储存在 4°C 下数天,或在 -20°C 冷冻,以延长存储时间。</li>
	<li>将40μL蛋白酶K制备到1.5 mL微离心?...

<ol>
	<li>Drachenberg, C. B., 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=Histological+patterns+of+polyomavirus+nephropathy:+correlation+with+graft+outcome+and+viral+load.">Histological patterns of polyomavirus nephropathy: correlation with graft outcome and viral load.</a> American Journal of Transplant. 4 (12), 2082-2092 (2004).</li><li>Korth, 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=Impact+of+low-level+BK+polyomavirus+viremia+on+intermediate-term+renal+allograft+function.">Impact of low-level BK polyomavirus viremia on intermediate-term renal allograft function.</a> Transplant Infectious Disease. 20 (1), (2018).</li><li>Hirsch, H. 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=European+perspective+on+human+polyomavirus+infection,+replication+and+disease+in+solid+organ+transplantation.">European perspective on human polyomavirus infection, replication and disease in solid organ transplantation.</a> Clinical Microbiology and Infection. 20 Suppl 7, 74-88 (2014).</li><li>Masutani, K., 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=The+Banff+2009+Working+Proposal+for+polyomavirus+nephropathy:+a+critical+evaluation+of+its+utility+as+a+determinant+of+clinical+outcome.">The Banff 2009 Working Proposal for polyomavirus nephropathy: a critical evaluation of its utility as a determinant of clinical outcome.</a> American Journal of Transplantation. 12 (4), 907-918 (2012).</li><li>Korth, 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=Impact+of+immune+suppressive+agents+on+the+BK-Polyomavirus+non+coding+control+region.">Impact of immune suppressive agents on the BK-Polyomavirus non coding control region.</a> Antiviral Research. 159, 68-76 (2018).</li><li>Hirsch, H. H., Yakhontova, K., Lu, M., Manzetti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BK+Polyomavirus+Replication+in+Renal+Tubular+Epithelial+Cells+Is+Inhibited+by+Sirolimus,+but+Activated+by+Tacrolimus+Through+a+Pathway+Involving+FKBP-12.">BK Polyomavirus Replication in Renal Tubular Epithelial Cells Is Inhibited by Sirolimus, but Activated by Tacrolimus Through a Pathway Involving FKBP-12.</a> Amerian Journal of Transplantation. 16 (3), 821-832 (2016).</li><li>Sanchez Fructuoso, A. 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=Mammalian+target+of+rapamycin+signal+inhibitors+could+play+a+role+in+the+treatment+of+BK+polyomavirus+nephritis+in+renal+allograft+recipients.">Mammalian target of rapamycin signal inhibitors could play a role in the treatment of BK polyomavirus nephritis in renal allograft recipients.</a> Transplant Infectious Disease. 13 (6), 584-591 (2011).</li><li>Moens, U., Johansen, T., Johnsen, J. I., Seternes, O. M., Traavik, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncoding+control+region+of+naturally+occurring+BK+virus+variants:+sequence+comparison+and+functional+analysis.">Noncoding control region of naturally occurring BK virus variants: sequence comparison and functional analysis.</a> Virus Genes. 10 (3), 261-275 (1995).</li><li>Gosert, R., 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=Polyomavirus+BK+with+rearranged+noncoding+control+region+emerge+in+vivo+in+renal+transplant+patients+and+increase+viral+replication+and+cytopathology.">Polyomavirus BK with rearranged noncoding control region emerge in vivo in renal transplant patients and increase viral replication and cytopathology.</a> Journal of Experimental Medicine. 205 (4), 841-852 (2008).</li><li>Bethge, T., 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=Sp1+sites+in+the+noncoding+control+region+of+BK+polyomavirus+are+key+regulators+of+bidirectional+viral+early+and+late+gene+expression.">Sp1 sites in the noncoding control region of BK polyomavirus are key regulators of bidirectional viral early and late gene expression.</a> Journal of Virology. 89 (6), 3396-3411 (2015).</li><li>Gosert, R., Kardas, P., Major, E. O., Hirsch, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearranged+JC+virus+noncoding+control+regions+found+in+progressive+multifocal+leukoencephalopathy+patient+samples+increase+virus+early+gene+expression+and+replication+rate.">Rearranged JC virus noncoding control regions found in progressive multifocal leukoencephalopathy patient samples increase virus early gene expression and replication rate.</a> Journal of Virology. 84 (20), 10448-10456 (2010).</li><li>Bernhoff, E., Gutteberg, T. J., Sandvik, K., Hirsch, H. H., Rinaldo, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cidofovir+inhibits+polyomavirus+BK+replication+in+human+renal+tubular+cells+downstream+of+viral+early+gene+expression.">Cidofovir inhibits polyomavirus BK replication in human renal tubular cells downstream of viral early gene expression.</a> American Journal of Transplantation. 8 (7), 1413-1422 (2008).</li><li>Widera, 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=HIV-1+persistent+viremia+is+frequently+followed+by+episodes+of+low-level+viremia.">HIV-1 persistent viremia is frequently followed by episodes of low-level viremia.</a> Medical Microbiology Immunology. , (2017).</li><li>Zhang, S., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purifying+plasmid+DNA+from+bacterial+colonies+using+the+QIAGEN+Miniprep+Kit.">Purifying plasmid DNA from bacterial colonies using the QIAGEN Miniprep Kit.</a> Journal of Visualized Experiment. (6), 247 (2007).</li><li>Drew, R. J., Walsh, A., Laoi, B. N., Crowley, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+analysis+of+the+complete+genome+of+11+BKV+isolates+obtained+from+allogenic+stem+cell+transplant+recipients+in+Ireland.">Phylogenetic analysis of the complete genome of 11 BKV isolates obtained from allogenic stem cell transplant recipients in Ireland.</a> Journal of Medical Virology. 84 (7), 1037-1048 (2012).</li><li>Dorries, K., Vogel, E., Gunther, S., Czub, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+human+polyomaviruses+JC+and+BK+in+peripheral+blood+leukocytes+from+immunocompetent+individuals.">Infection of human polyomaviruses JC and BK in peripheral blood leukocytes from immunocompetent individuals.</a> Virology. 198 (1), 59-70 (1994).</li><li>Teutsch, K., 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=Early+identification+of+renal+transplant+recipients+with+high+risk+of+polyomavirus-associated+nephropathy.">Early identification of renal transplant recipients with high risk of polyomavirus-associated nephropathy.</a> Medical Microbiology Immunology. 204 (6), 657-664 (2015).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+specific+cell+population+by+fluorescence+activated+cell+sorting+(FACS).">Purification of specific cell population by fluorescence activated cell sorting (FACS).</a> Journal of Visualized Experiment. (41), (2010).</li><li>Korth, 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=The+detection+of+BKPyV+genotypes+II+and+IV+after+renal+transplantation+as+a+simple+tool+for+risk+assessment+for+PyVAN+and+transplant+outcome+already+at+early+stages+of+BKPyV+reactivation.">The detection of BKPyV genotypes II and IV after renal transplantation as a simple tool for risk assessment for PyVAN and transplant outcome already at early stages of BKPyV reactivation.</a> Journal of Clinical Virology. 113, 14-19 (2019).</li><li>Caputo, A., Barbanti-Brodano, G., Wang, E., Ricciardi, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transactivation+of+BKV+and+SV40+early+promoters+by+BKV+and+SV40+T-antigens.">Transactivation of BKV and SV40 early promoters by BKV and SV40 T-antigens.</a> Virology. 152 (2), 459-465 (1986).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nature Methods. 2 (12), 905-909 (2005).</li></ol>

本文提出了一种常用的方法,用于分析BKPyV非编码控制区域(NCCR)驱动的早期和后期启动器活动。NCCR活性可以同时测量,不需要转染细胞的溶解。此外,可以分析相对大量的细胞,并且不需要共同转染额外的标记,以使荧光值正常化。
此方法的一个关键部分是,克隆的 NCCR 应包含与阿普利森测序所识别的完全相同的序列,该序列对应于患者血浆中存在的大多数变体。为了获得准确的结果?...

聚瘤病毒代表一个独立的小双链DNA(dsDNA)病毒家族,以西米安病毒40(SV40)为原型物种。原发性感染主要发生在儿童期,通常无疾病症状,通常引起免疫宿主的潜在感染。BK-多瘤病毒(BKPyV)主要在肾小管细胞中持续存在,不会引起肾病,但是,在肾移植后免疫能力受损的情况下,病毒可以重新激活,并造成严重的损害和受损的移植物功能达到高病毒血症 (1 x 104 BKPyV DNA 拷贝/mL)1,2 。在大约10%的肾脏移植接受者中,BK-多瘤病毒(BKPyV)的重新激活导致多瘤病毒相关肾病(PyVAN),高达80%与肾异体移植失败的高风险相关3,4.由于没有经过批准的抗病毒药物,目前的治疗是基于减少免疫抑制。有趣的是,mTOR抑制剂似乎具有抗病毒作用;因此,将免疫抑制疗法切换到基于mTOR的免疫抑制可能是防止BKPyV病毒血症5、6、7进展的替代方法。然而,基于mTOR的抗病毒机制目前仍不完全了解。因此,需要测量潜在抗病毒药物在临床相关浓度中的影响的方法。
BKPyV的循环基因组包括大约5 kb,包含一个非编码控制区域(NCCR),作为复制的起源,并同时作为一个双向启动子,推动早期和晚期相mRNA转录的表达。由于自发发生的NCCR-重排、删除和复制在致病性BKPyV8中发现,并且明显累积在PVAN 5,9患者中,原型(wt)和重新排列 (rr) NCCR 活动有助于描述病毒复制性适应性。
如图1所概述,该协议描述了一种常用的方法,通过量化从报告质粒5表达的两种荧光团和eGFP的荧光来测量BKPyV NCCR转录活性。 9,10,11.该程序在SV40大T抗原(lTAg)的存在下进行,它允许分析潜在的抗病毒药物对NCCR活性的早期和晚期的影响5。该测定进一步分析了重组对NCCR活动的影响,并与wt-NCCRs5、9进行了比较。记者将SV40晚聚光信号下行到每个荧光光片开放读取帧的下游,以确保分别对tdTomato和eGFP的两种转录本进行可比和高效的处理。与基于qRT-PCR的方法5,12相比,这种基于FACS的方法代表了低成本和高吞吐量兼容的替代方法,因为没有复杂的提取协议,感染细胞培养,没有昂贵的需要用于免疫荧光染色的抗体。此外,由于通过流式细胞测定分析定义的荧光细胞量,因此也可以定量分析细胞周期抑制剂。

<table border="0" cellpadding="0" cellspacing="0" style="width:959px;" width="957">
	<tbody>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">100 bp ladder</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">N3231</td>
			<td style="width:379px;">Any ladder with a range up to 1 kb can be substituted</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">2-log ladder</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">N0550</td>
			<td>Any ladder with a range up to 10 kb can be substituted</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Agar-Agar, Kobe I</td>
			<td style="width:127px;">Carl Roth</td>
			<td style="width:151px;">5210.3</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">AgeI-HF</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">R3552</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Ampicillin Natriumsalz Cellpure</td>
			<td style="width:127px;">Carl Roth</td>
			<td style="width:151px;">HP62.1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Aqua ad iniectabilia</td>
			<td style="width:127px;">Bbraun</td>
			<td style="width:151px;">2351744</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">BD FACSCanto&#8482; II</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">BD FACSDiva</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">DAPI</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:151px;">10236276001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">DFC450C camera module</td>
			<td style="width:127px;">Leica</td>
			<td style="width:151px;"></td>
			<td style="width:379px;">Any camera can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">DMEM</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:151px;">41966-029</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">DMIL LED microscope</td>
			<td style="width:127px;">Leica</td>
			<td style="width:151px;"></td>
			<td style="width:379px;">Any fluorescence microscope can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">DNA Blood Mini Kit</td>
			<td style="width:127px;">Qiagen</td>
			<td style="width:151px;">51104</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">E.coli DH5alpha Competent Cells</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td style="width:151px;">18258012</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">FBS Superior</td>
			<td style="width:127px;">MerckMillipore</td>
			<td style="width:151px;">50615</td>
			<td style="width:379px;">Any FBS can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">FlowJo v10.5.3</td>
			<td style="width:127px;">FlowJo, LLC</td>
			<td style="width:151px;"></td>
			<td style="width:379px;">Any flow cytometry software FBS can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Gel Loading Dye, Purple (6X)&#160;</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">B7024</td>
			<td>Any 6x loading dye can be substituted</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">HEK293T cells</td>
			<td style="width:127px;">ATCC</td>
			<td style="width:151px;">11268</td>
			<td>These cells constitutively express the simian virus 40 (SV40) large T antigen, and clone 17 was selected specifically for its high transfectability.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">HERAcell&#174; 240i CO2 Incubator</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td style="width:151px;"></td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">HotStar PCR kit</td>
			<td style="width:127px;">Qiagen</td>
			<td style="width:151px;">203203</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Intas Gel documentation system</td>
			<td style="width:127px;">Intas</td>
			<td style="width:151px;"></td>
			<td>Any visualisation system for stained DNA containing agarose gels can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Low Melt Agarose</td>
			<td style="width:127px;">Biozym</td>
			<td style="width:151px;">850081</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Opti-MEM</td>
			<td style="width:127px;">Invitrogen</td>
			<td style="width:151px;">31985070</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">pBKV (34-2)</td>
			<td style="width:127px;">ATCC</td>
			<td style="width:151px;">45025</td>
			<td>Plasmid harboring the full-length genome of BKPyV strain Dunlop; was used as a positive control; DNA Seq. Acc.: KP412983</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">PBS</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:151px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">PCR Cycler MJ Mini 48-Well Personal Cycler</td>
			<td style="width:127px;">Bio-Rad</td>
			<td style="width:151px;">discontinued product</td>
			<td style="width:379px;">Any thermocycler can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">PCR Nucleotide Mix, 10 mM</td>
			<td style="width:127px;">Promega</td>
			<td style="width:151px;">#C1145</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">PCR1 and PCR2 Primers</td>
			<td style="width:127px;">metabion</td>
			<td style="width:151px;">not applicable</td>
			<td>Desalted. Dilute to (10 &#181;M) with PCR grade water</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">PenStrep (100x)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:151px;">15140-122</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Roti&#174;-GelStain</td>
			<td style="width:127px;">Carl Roth</td>
			<td style="width:151px;">3865</td>
			<td>A fluorescence based stain for measuring dsDNA concentration</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">SpeI-HF</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">R3133</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">T4-DNA Ligase</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">M0202</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">TransIT LT1</td>
			<td style="width:127px;">Mirus</td>
			<td style="width:151px;">MIR2300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Trypsin 0.05% - EDTA</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:151px;">25300-054</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">ZymoPURE II Plasmid Kit</td>
			<td style="width:127px;">Zymo</td>
			<td style="width:151px;">D4201</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
	</tbody>
</table>

measurement of bk-polyomavirus non-coding control region driven transcriptional activity via flow cytometry

多瘤病毒,如BK-多马病毒(BKPyV),可引起免疫功能低下患者的严重疾病。然而,由于目前没有高效抗病毒药物,需要用测量潜在抗病毒药物影响的方法。在这里,一个双荧光报告器,允许分析BKPyV非编码控制区域(NCCR)驱动的早期和后期促进者活动,以量化潜在的抗病毒药物对病毒基因表达的影响,通过tdTomato和eGFP表达。此外,通过克隆BKPyV-NCCR增压,该议定书中从免疫功能低下的肾移植患者的血液衍生DNA中得到的,可以确定NCCR-重组对病毒基因表达的影响。在克隆患者衍生的氨基细胞后,HEK293T细胞与报告质粒转染,并用潜在的抗病毒剂进行治疗。随后,对细胞进行FACS分析,以测量转染后72小时的平均荧光强度。为了测试具有潜在细胞循环抑制作用的药物的分析,只使用转染的荧光细胞。由于这种测定是在大T抗原表达细胞中进行的,因此可以相互独立地分析早期和晚期表达的影响。

在具有代表性的实验中,通过流式细胞测定测量了BK-多瘤病毒非编码控制区驱动的转录活性。此外,一种mTOR抑制剂,可用于BKPyV重新激活后治疗患者,被测试为抑制病毒早期基因表达。为此,使用双荧光-报告器测定,如之前公布的5。实验设置的总体工作流程方案在F igure 1中进行了说明。首先,根据机构伦理委员会批准的人类研究指南,从?...

Watch this Scientific Journal Video about Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry at JoVE.com

Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry

BK-多瘤病毒非编码控制区域驱动转录活性通过流细胞测定的测量

在本手稿中,提出了使用 HEK293T 细胞转染的基于 FACS 的 BK-多瘤病毒转录活性的测量方案,该细胞与表达 tdTomato 和 eGFP 的双向报告质粒转染。该方法进一步允许定量确定新化合物对病毒转录的影响。

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals ...

该协议的总体目标是通过流式细胞测量双荧光细胞，测量BK-多病毒非编码控制区域驱动的转录活性。BK-多瘤病毒可导致免疫功能低下的患者出现严重疾病，尤其是肾移植接受者。然而，由于目前没有有效的杀毒软件，因此需要测量其他化合物的潜在抗病毒影响的方法。这种方法允许病毒学家分析由非编码控制区域驱动的BK-多孔病毒转录活动的潜在抗病毒剂的影响。该分析进一步允许研究重新排列对启动子活动的影响，与典型的非编码控制区域相比。用双荧光报告研究病毒促进活动的好处是，可以相互独立地分析早期和晚期基因表达。此外，由于只分析转染和荧光细胞，因此不需要对病毒储存的困难穿孔和长期感染细胞培养实验进行分析，还可以研究降低细胞生存能力和穿孔的药物。该协议遵循杜伊斯堡-埃森大学医学院伦理委员会批准的人类研究指南。第一步是收集血液样本，以分离多瘤病毒DNA。在EDTA采集管中收集至少三毫升的血液。将样品在2，500G下离心15分钟，将等离子体移液到新管中。将40微升蛋白质SK放入1.5毫升微离心管中，通过移液加入400微升等离子体。使用制造商说明中所述的 DNA 污点提取试剂盒分离 DNA。使用总体积为 50 微升的底向对 A 为 PCR 准备主组合，并使用先前分离的 DNA。将主混合物的 45 微升分配到 PCR 管中。将分离的DNA的五微升加入PCR管中，并在反应条件下运行PCR，如表三所示。在 35 个周期内重复变性退火和延长。对于嵌套应用程序，请使用底像对 B，并包含用于克隆的限制站点。将 PCR 产品的 10 微升与两个六折凝胶加载染料的微升混合。在 1.5% 的加糖凝胶上装载 10 微升混合物，并在 60 毫安培下运行凝胶 30 分钟。使用适当的紫外线记录系统可视化凝胶。使用 PCR 纯化套件根据制造商的说明进行纯化 PCR 安普利。在37摄氏度下用指标限制酶消化纯化的安普利森两小时。重复净化步骤以净化消化的安普利孔。同时，还消化质粒骨干。在0.8%的低安装加糖凝胶上分析消化的质粒骨干。请勿在高于 40 毫安的电流下运行凝胶。使用长波紫外光可视化DNA片段，并使用干净的手术刀切割骨干弯曲。避免长时间的紫外线照射，防止DNA损伤。将一块浮糖凝胶碎片放入新的1.5毫升微离心管的围栏。在65摄氏度下加热含有低模气糖片的骨干10分钟，以熔化凝胶片，每两分钟通过轻轻的涡流混合一次。熔化的凝胶可直接用于结扎。利盖特骨干和消化的安培康使用T4 DNA连接酶过夜在16摄氏度。经过标准克隆程序，分离血浆DNA。执行限制性酶消化，检查阳性克隆，并在1%的阿加罗斯凝胶上可视化消化片段。种子100，000 HEK293T细胞每井12井板前24小时转染和孵育过夜在37摄氏度，保持活增殖在转染期间。细胞在转染时应大约在80%的汇合。将250微升的减少血清介质放在无菌管中，并加入每组报告的血浆DNA一微克，通过移液轻轻混合。将转染试剂的三微升加入脱氧核糖核酸混合物中，通过移液和孵育在22摄氏度下轻轻混合15分钟。加入混合物，滴到井中，轻轻分配到井中。四小时后，用含有测试剂和溶剂控制的新鲜介质更换上一液。在此示例中，使用了 mTOR 抑制剂 INK-128 和拉帕霉素。在37摄氏度下孵育，直到分析。在荧光显微镜下检查细胞有红荧光和绿色荧光。红色和绿色荧光分别对应于早期和晚期BK-多病毒基因表达。转染后 72 小时，精心向往上一道。用一毫升的冷PBS清洗细胞两次。轻轻加入500微升的试金，稍微转动板，避免细胞过早分离。此步骤对于避免单元格双精度很重要。添加后，使用相同的移液器尖端直接去除三辛。在37摄氏度下孵育细胞至少5分钟。用含有 3%FCS 的毫升 PBS 重新悬浮 trypsin S 细胞，并在预标记的 FACS 管中转移悬浮液。在 FACS 分析之前，以每毫升一微克的浓度添加 DAPI。使用流动细胞计分析细胞。对于每个样本，测量至少10，000个活细胞。初始补偿对于区分红色和绿色信号以实现准确结果至关重要。使用嵌套 PCR 协议放大非编码控制摄政，该协议使用内部底像对进行放大，该底的是包含克隆限制站点的。安培孔验证是通过阿加罗斯凝胶电泳进行的，而从典型的NCCR序列中派生的安培子在凝胶中具有同质大小分布，而从重新排列的NCCR中派生的带插入和删除的NCCR，其大小不同。通过限制分析验证了含有NCCR的正克隆的选择。小空间区域被指示正克隆的较大 NCCR 片段所取代。从经过验证的克隆中分离出的质粒DNA被送去进行脱氧核糖。在此示例中，检测到 P 块中的删除和 O 块中的替换。HEK293T细胞在尊重可报告结构时瞬态转染，NCCR活性通过荧光显微镜进行监测。红色荧光和绿色荧光分别对应于早期和晚期BK-多病毒基因表达。第一个NCCR显示强后期基因表达，而第二个例子具有相对强的早期但中度晚基因表达。DAPI 负细胞被封口，并创建了一个点图，显示 eGFP 与 tdTomato。仅包含阴性样本以及 tdTomato 或 eGFP 阳性样本。平均荧光强度来自每个测试克隆。在此示例中，使用双 mTOR 抑制剂 INK128 的治疗显著减少了 tdTomato MFI，这意味着早期表达被抑制。此方法允许通过转录活性识别当前使用的其他化合物来检测 BK-多瘤病毒。在早期的基因表达中，这决定了病毒的含意。在BK-多孔病毒重新激活的情况下，抑制滴可被视为免疫功能低下患者的剂。

Research

JoVE Journal

Genetics

Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma

经典霍奇金淋巴瘤的芦苇细胞的流分选和外显子测序

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically ...

科研

遗传学

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

rna 下拉法识别长非编码 rna 的 rna 靶点

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

基于细胞的 sineup 非编码 rna 检测, 可专门增强 mrna 的翻译能力

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. ...

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

利用 niper-cas9 在不损失目标活动的情况下通过定向进化, 最大限度地减少 crispr-cas9 的非目标效应

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

remtoe 控制系统在内源基因表达调控中的应用

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

用于抗倍体植入前基因测试的半导体测序

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

一种通用嵌套逆转录酶链反应检测裂解病毒的评价

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

用于分离共转录RNA处理事件的人工RNA聚合酶II伸长复合物

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...