Name: methods to discover alternative promoter usage and transcriptional regulation of murine bcrp1
Uploaded: 2016-05-27T14:00:00
Description: Watch this Scientific Journal Video about Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1 at JoVE.com

这项工作得到了优异评论奖道格拉斯D.罗斯和从退伍军人事务部的阿里夫·侯赛因的支持。

1. 在 BCRP1的替代外显子第一硅片预测上使用鼠标EST数据库的BLAST分析注：本协议描述了如何搜索小鼠表达序列标签（EST）数据库与序列相似性的无害环境技术外显子BCRP1（包含翻译起始位点）2，再怎么匹配的EST序列对准基因组序列，以确定他们的位置相对于BCRP1外显子2的5&#39;末端的小鼠BCRP1基因英寸<ol><li>通过输入mRNA的参考序列ID（NM_011920.3）进入ENSEMBL基因组浏...

<ol>
	<li>Kimura, 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=Diversification+of+transcriptional+modulation:+large-scale+identification+and+characterization+of+putative+alternative+promoters+of+human+genes.">Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.</a> Genome Res. 16 (1), 55-65 (2006).</li><li>Doyle, L. 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=A+multidrug+resistance+transporter+from+human+MCF-7+breast+cancer+cells.">A multidrug resistance transporter from human MCF-7 breast cancer cells.</a> Proc Natl Acad Sci U S A. 95 (26), 15665-15670 (1998).</li><li>Natarajan, K., Xie, Y., Baer, M. R., Ross, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+breast+cancer+resistance+protein+(BCRP/ABCG2)+in+cancer+drug+resistance.">Role of breast cancer resistance protein (BCRP/ABCG2) in cancer drug resistance.</a> Biochem Pharmacol. 83 (8), 1084-1103 (2012).</li><li>Qian, X., Cheng, Y. H., Mruk, D. D., Cheng, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+resistance+protein+(Bcrp)+and+the+testis--an+unexpected+turn+of+events.">Breast cancer resistance protein (Bcrp) and the testis--an unexpected turn of events.</a> Asian J Androl. 15 (4), 455-460 (2013).</li><li>Xie, Y., 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=Bcrp1+transcription+in+mouse+testis+is+controlled+by+a+promoter+upstream+of+a+novel+first+exon+(E1U)+regulated+by+steroidogenic+factor-1.">Bcrp1 transcription in mouse testis is controlled by a promoter upstream of a novel first exon (E1U) regulated by steroidogenic factor-1.</a> Biochim Biophys Acta. 1829 (12), 1288-1299 (2013).</li><li>Maliepaard, 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=Subcellular+localization+and+distribution+of+the+breast+cancer+resistance+protein+transporter+in+normal+human+tissues.">Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues.</a> Cancer Res. 61 (8), 3458-3464 (2001).</li><li>Fetsch, P. 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=Localization+of+the+ABCG2+mitoxantrone+resistance-associated+protein+in+normal+tissues.">Localization of the ABCG2 mitoxantrone resistance-associated protein in normal tissues.</a> Cancer Lett. 235 (1), 84-92 (2006).</li><li>Cooray, H. C., Blackmore, C. G., Maskell, L., Barrand, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localisation+of+breast+cancer+resistance+protein+in+microvessel+endothelium+of+human+brain.">Localisation of breast cancer resistance protein in microvessel endothelium of human brain.</a> Neuroreport. 13 (16), 2059-2063 (2002).</li><li>Kolwankar, D., Glover, D. D., Ware, J. A., Tracy, T. S. <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+function+of+ABCB1+and+ABCG2+in+human+placental+tissue.">Expression and function of ABCB1 and ABCG2 in human placental tissue.</a> Drug Metab Dispos. 33 (4), 524-529 (2005).</li><li>Xiong, 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=ABCG2+is+upregulated+in+Alzheimer's+brain+with+cerebral+amyloid+angiopathy+and+may+act+as+a+gatekeeper+at+the+blood-brain+barrier+for+Abeta(1-40)+peptides.">ABCG2 is upregulated in Alzheimer's brain with cerebral amyloid angiopathy and may act as a gatekeeper at the blood-brain barrier for Abeta(1-40) peptides.</a> J Neurosci. 29 (1-40), 5463-5475 (2009).</li><li>Woodward, O. 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=Identification+of+a+urate+transporter,+ABCG2,+with+a+common+functional+polymorphism+causing+gout.">Identification of a urate transporter, ABCG2, with a common functional polymorphism causing gout.</a> Proc Natl Acad Sci U S A. 106 (25), 10338-10342 (2009).</li><li>Huls, 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=The+breast+cancer+resistance+protein+transporter+ABCG2+is+expressed+in+the+human+kidney+proximal+tubule+apical+membrane.">The breast cancer resistance protein transporter ABCG2 is expressed in the human kidney proximal tubule apical membrane.</a> Kidney Int. 73 (2), 220-225 (2008).</li><li>Nakanishi, 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=Novel+5'+untranslated+region+variants+of+BCRP+mRNA+are+differentially+expressed+in+drug-selected+cancer+cells+and+in+normal+human+tissues:+implications+for+drug+resistance,+tissue-specific+expression,+and+alternative+promoter+usage.">Novel 5' untranslated region variants of BCRP mRNA are differentially expressed in drug-selected cancer cells and in normal human tissues: implications for drug resistance, tissue-specific expression, and alternative promoter usage.</a> Cancer Res. 66 (10), 5007-5011 (2006).</li><li>Ayoubi, T. A., Van De Ven, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+gene+expression+by+alternative+promoters.">Regulation of gene expression by alternative promoters.</a> FASEB J. 10 (4), 453-460 (1996).</li><li>Zong, Y., Zhou, S., Fatima, S., Sorrentino, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+mouse+Abcg2+mRNA+during+hematopoiesis+is+regulated+by+alternative+use+of+multiple+leader+exons+and+promoters.">Expression of mouse Abcg2 mRNA during hematopoiesis is regulated by alternative use of multiple leader exons and promoters.</a> J Biol Chem. 281 (40), 29625-29632 (2006).</li><li>Natarajan, 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=Identification+and+characterization+of+the+major+alternative+promoter+regulating+Bcrp1/Abcg2+expression+in+the+mouse+intestine.">Identification and characterization of the major alternative promoter regulating Bcrp1/Abcg2 expression in the mouse intestine.</a> Biochim Biophys Acta. 1809 (7), 295-305 (2011).</li><li>Promega. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Wizard&#174; SV Gel and PCR Clean-Up System - INSTRUCTIONS FOR USE OF PRODUCT. , (2009).</li><li>New_England_Biolabs. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quick Ligation Kit Protocol - M2200S. , (2014).</li><li>Roche. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> XtremeGENE HP DNA Transfection Reagent Protocol - Manual version 1.0). , (2010).</li><li>Promega. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quick Protocol for the use of the Dual-Luciferase Reporter Assay. , (2009).</li><li>Carninci, P., 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+transcriptional+landscape+of+the+mammalian+genome.">The transcriptional landscape of the mammalian genome.</a> Science. 309 (5740), 1559-1563 (2005).</li><li>VanBuren, V., 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=Assembly,+verification,+and+initial+annotation+of+the+NIA+mouse+7.4K+cDNA+clone+set.">Assembly, verification, and initial annotation of the NIA mouse 7.4K cDNA clone set.</a> Genome Res. 12 (12), 1999-2003 (2002).</li><li>Bonaldo, M. F., Lennon, G., Soares, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+and+subtraction:+two+approaches+to+facilitate+gene+discovery.">Normalization and subtraction: two approaches to facilitate gene discovery.</a> Genome Res. 6 (9), 791-806 (1996).</li><li>Okazaki, Y., 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+the+mouse+transcriptome+based+on+functional+annotation+of+60,770+full-length+cDNAs.">Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs.</a> Nature. 420 (6915), 563-573 (2002).</li><li>Landry, J. R., Mager, D. L., Wilhelm, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+controls:+the+role+of+alternative+promoters+in+mammalian+genomes.">Complex controls: the role of alternative promoters in mammalian genomes.</a> Trends Genet. (11), 640-648 (2009).</li><li>Park, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq:+advantages+and+challenges+of+a+maturing+technology.">ChIP-seq: advantages and challenges of a maturing technology.</a> Nat Rev Genet. 10 (10), 669-680 (2009).</li><li>Bulun, S. E., 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=Regulation+of+aromatase+expression+in+breast+cancer+tissue.">Regulation of aromatase expression in breast cancer tissue.</a> Ann N Y Acad Sci. 1155, 121-131 (2009).</li></ol>

大多数提出的方法和代表性的结果在以前的题目这是发表在2013年5“在小鼠睾丸乙 CRP1转录由启动子上游通过类固醇生成因子-1调控的小说第一外显子（E1U），可控”的工作介绍除了这里所描述的代表性的结果，先前的纸张提供了一种使用5&#39;-RACE PCR和RT-PCR检测方法替代第一外显子的利用率的估计。此外，在硅片在启动子上游E1U推定SF-1应答元件的鉴定来完成，和染色质免疫沉?...

超过人类基因中有一半是通过促 ​​进替代上调1。每个备选启动子可以含有调控元件，可能是从那些在其他替代的启动子不同。在一个组织中使用的启动子（多个）可能与另一组织中使用不同。例如，它可能是一个给定的信号传导途径的激活可以触发用于在一个组织利用的基因替代的启动子，但有没有影响或抑制对于被利用在另一组织相同基因的单独的替代型启动子。 所述BCRP1基因的表达是通过替代的启动子支配。 BCRP1是人类乳腺癌耐药蛋白（BCRP）基因的小鼠同源基因。 BCRP是ATP结合盒（ABC）转运，正式指定ABCG2 2，3。作为一个顶质膜蛋白质，BCRP / BCRP1功能外排多种天然和异生底物<suP&gt; 3，4。在人类和小鼠中，BCRP / BCRP1高度在药理学相关机关表示如肝（胆小管），肠和肾，以及器官血液-组织屏障如胎盘，脑和睾丸2，5 - 12。 BCRP / BCRP1的造血和其它干细胞包括癌干细胞，表达可以赋予这些细胞的抗外源物和癌症化疗药物3。 在早期的工作，了解BCRP表达在正常和肿瘤细胞的调节，进行5&#39;cDNA末端（5&#39;-RACE）BCRP mRNA的分析，快速扩增，以确定其确切的转录起始位点13。不仅是找到多个转录起始位点;也遇到是第一外显子，这是BCRP翻译三种替代形式。这些替代第一外显子 - 指定中E1a，E1B，E1C - 在各种人体组织中表达不同。两个附加第一外显子的变体在BLAST搜索用BCRP 13的第二外显子的人的EST数据库的被发现。四场比赛显露上游从被指定E1u外显子2第一外显子&gt; 70 kb的;其他四场比赛显露BCRP表达，缺乏完全是第一外显子，这被指定E1- 13。 替代领导人外显子的存在被认为是替代的启动子的使用14的体现。 在小鼠中，BCRP1四个替代第一外显子中描述了可以对应于管理在不同鼠组织BCRP 1转录替代的启动子;这些外显子/启动子指定E1U，E1A，E1B和E1C，并位于约70，从BCRP1外显子2 5,15 58，15和5 kb的上游。在E1A的mRNA同种型被认为是在murin主要È造血干细胞，心脏，肺，脾和脑，而E1B亚型在小鼠小肠，胎肝细胞和红细胞前体细胞在骨髓5,15表示。上游E1B启动子被证明是管辖在小鼠肠BCRP1转录的主要替代子，至少部分调节的，由磷酸环-AMP反应元件结合蛋白（对CREB）和独特的，替代一个CREB响应元件BCRP1子16。该E1C表达亚型在成年小鼠肝脏和肾脏5中表达。该E1U亚型是在除了鼠睾丸，它是表示5的主要同种型测试大多数组织中检测不到。 BCRP1在大鼠睾丸表达的在两个躯体（内皮细胞紧密连接，管周肌样细胞和支持细胞）细胞和生殖细胞中发现（生精内皮细胞，它可以保护晚期精子4）。该注册离子上游E1U包含类固醇生成因子1（SF-1）5的官能响应元素。 BCRP1 mRNA和蛋白在塞尔托利细胞特异性SF-1敲除小鼠的睾丸被显着降低，这表明在小鼠Sertoli细胞BCRP1表达通过SF-1 5控制。 详细介绍方法的协议来检测BCRP1替代第一外显子在硅片上 ，并从上游确定的替代外显子第一次为建立促进推测基于荧光素酶报告基因分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>COMMERCIAL BIOLOGIC MATERIALS AND KITS:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>First Choice RLM-RACE kit</td>
			<td>Ambion Inc., Austin, TX, currently available through Life Technologies</td>
			<td>AM1700</td>
			<td>5&#39;-RACE PCR kit</td>
		</tr>
		<tr>
			<td>TOPO TA cloning kit&#160;</td>
			<td>Life Technologies</td>
			<td>K450001</td>
			<td>This kit contains the TOP10 chemically competent E. coli bacteria.</td>
		</tr>
		<tr>
			<td>T4 DNA ligase quick ligation kit</td>
			<td>New England Biolabs</td>
			<td>M2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Faststart high-fidelity Taq- DNA polymerase</td>
			<td>Sigma-Aldrich/Roche</td>
			<td>3553400001/RMB-4738284001&#160;</td>
			<td>Contains a high-fidelity Taq polymerase enzyme blend</td>
		</tr>
		<tr>
			<td>XtremeGENE HP DNA transfection reagent</td>
			<td>Roche</td>
			<td>6366236001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-luciferase reporter assay kit</td>
			<td>Promega</td>
			<td>E1910</td>
			<td>Includes the pGL3-basic empty reporter vector (firefly luciferase), pRLTK renilla luciferase expressing vector, and other control vectors.</td>
		</tr>
		<tr>
			<td>QIAprep</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>For plasmid miniprep - isolation and purification of plasmid DNA from bacterial colonies</td>
		</tr>
		<tr>
			<td>pCR2.1 vector</td>
			<td>part of the TOPO TA cloning kit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pGL3-basic luciferase containing vector</td>
			<td>Promega</td>
			<td>E1751</td>
			<td></td>
		</tr>
		<tr>
			<td>pRL-TK Renilla luciferase expressing vector</td>
			<td>Promega</td>
			<td>E2241</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial artificial chromosome</td>
			<td>BACPAC Resources Center, Children&#8217;s Hospital Oakland Research Institute, Oakland, CA</td>
			<td>RP23-285A12 and RP24-314E24</td>
			<td>http://bacpac.chori.org/clones.htm</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>REAGENTS/CHEMICALS/MEDIA/SUPPLIES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Life Technologies</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard&#174; SV Gel and PCR Clean-Up System&#160;</td>
			<td>Promega</td>
			<td>A9281</td>
			<td>Useful for purifying PCR products following digestion with restriction endonucleases</td>
		</tr>
		<tr>
			<td>CRYOVIALS</td>
			<td>Denville Scientific</td>
			<td>V9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>SOFTWARE:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ensembl software</td>
			<td>Ensembl project</td>
			<td>http://Ensembl.org&#160;</td>
			<td>The Ensembl project produces genome databases for vertebrates and other eukaryotic species, and makes this information freely available online.</td>
		</tr>
		<tr>
			<td>Blast software</td>
			<td>NCBI</td>
			<td>http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD= 
			Web&#38;PAGE_TYPE=BlastHome</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer 3 software</td>
			<td>Simgene</td>
			<td>http://simgene.com/Primer3</td>
			<td>Useful for designing primers for 5&#39;-RACE PCR</td>
		</tr>
		<tr>
			<td>NCBI Nucleotide database</td>
			<td>NCBI</td>
			<td>http://www.ncbi.nlm.nih.gov/nucleotide/</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>CELL LINES:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TM4 murine Sertoli cells</td>
			<td>ATCC, Manassas, Virginia</td>
			<td>CRL-1715&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>INSTRUMENTS:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Turner Biosystems</td>
			<td>TD-20/20</td>
			<td>This is a relatively inexpensive, manually operated luminometer. Automated systems are also available from the same manufacturer that utilize 96 well plates.</td>
		</tr>
		<tr>
			<td>NanoDrop spectrophotometers</td>
			<td>Thermo Scientific, Inc.</td>
			<td>NanoDrop 2000</td>
			<td>Spectrophotometer can use very small quantities of sample</td>
		</tr>
		<tr>
			<td>DU 800 UV/VIS spectrophotometer and Nucleic Acid Analysis II software</td>
			<td>BeckmanCoulter Inc, Fullerton, CA</td>
			<td>DU 800</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods to discover alternative promoter usage and transcriptional regulation of murine bcrp1

通常非翻译 - - 第一外显子在不同组织中的基因表达通常通过导致mRNA的合成具有独特的备选启动子控制。 BCRP1（ABCG2），ABC转运乳腺癌耐药蛋白（BCRP，ABCG2）的小鼠同源基因，具有由生产相应的四个备选外显子先指定至少有四种可供选择的发起人：E1U，E1A，E1B和E1C。这里， 在计算机芯片上的协议被提供给预测备选启动子用法BCRP1。此外，被描述报告基因检测方法，以产生替代的启动子报道构建并确定推定的启动子上游的所确定的替代第一外显子的功能。

BCRP 1 替代促进利用 的鉴定 在小鼠睾丸被领导者的外显子的分析 当在NCBI的EST数据库，使用在方案1中列出的步骤进行探测（2015年4月）中，EST序列发现，分别邻接在BCRP的5&#39;外显子2的端部1 mRNA 的示于表1，与它们在基因组的位置?...

Watch this Scientific Journal Video about Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1 at JoVE.com

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

方法发现小鼠BCRP1的另类用法子和转录调控

与鼠ABC转运BCRP1（ABCG2）作为一个例子， 在硅片呈现协议，以检测在小鼠组织中表达的基因的备选启动子的使用，并评价使用报告基因分析鉴定的备选启动子的功能。

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

The overall goal of this set of protocols is to detect alternative first exons in the mRNA of a gene such as Bcrp1 and to establish luciferase-based reporter assays for the putative promoters upstream of the alternative identified first exons. The in-silico methods used can help answer key questions in the transcription and regulation field such as how mRNA expression is controlled in different tissues. The main advantage of this technique is that it uses publicly available genetic data to identify alterative first exons in the mRNA of gene of interest, which implies alternative promoter usage.This method that prepares report constructs and conducts reporter assays can help answer key questions in the gene regulatory field such as the functional activity of a putative alternative promoters. To begin this procedure, obtain the sequence for mouse Bcrp1 exon 2 by inputting the mRNA reference sequence ID into the search window of the Ensembl genome browser and click Go.Next, select a full length sequence that contains 16 exons. Click the Download sequence option.Then, choose the FASTA format and click Preview. In the next screen, copy the preview sequence of exon 2 into the clipboard. Navigate to the blast homepage on the NCBI website.Select Mouse genome. Paste the exon 2 sequence from the clipboard into the query box. After that, select ESTs from the Database drop down.Optimize it for Highly similar sequences and then choose BLAST. When the BLAST run is complete, the results page will appear. Under the Descriptions subheading of the results page, select All, then Download, followed by FASTA in the drop down, and then choose Continue.A txt file will appear. Open and copy the entire file into the clipboard. To identify the location of the EST sequence that is five prime to exon 2 in the Bcrp1 gene, on the BLAST homepage under the Specialized BLAST subheading, select Align two sequences using BLAST.Paste the text file from the clipboard into the query box and enter 372099104 in the Subject Sequence box. Optimize it for Highly similar sequences under Program Selection and run BLAST. Once the results window appears, view the alignments graphically by clicking Graphics in the Alignments window.Use the right and left arrows and zoom to focus on Bcrp1/ABCG2 and the sequence alignments. Then, save the sequence alignments by selecting All in the Descriptions box. Under the Download drop down, select Hit Table and then click Continue.As the position of the five prime end of exon 2 corresponds to the nucleotide 58, 655, 638 in the chromosome six contig, designate this as plus one and then calculate the position of the partial sequences of each EST five prime to 58, 655, 638. A critical step in this procedure is the prediction of multiple first exons. The alternative promoters are hence identified here.Using the sequence of E1U obtained from the EST database, designate the first five prime nucleotide of E1U as plus one. Obtain a back clone of mouse chromosome six that contains the Bcrp1/ABCG2 gene sequence and the sequence at least 100 kilobases upstream of the Bcrp1/ABCG2 gene. Then, identify a suitable reporter vector, such as the luciferase reporter plasmid pGL3-Basic.Determine the multiple cloning sites in the reporter vector. KpnI, SacI, MluI, NheI, SmaI, XhoI, BglII, and HindIII are the restriction endonucleae sites in the multiple cloning site of the pGL3-Basic vector in the five prime to three prime orientation. Next, select the two kilobase region upstream of the E1U exon and paste the sequence in the DNA5 followed by running the program.The details of the single and multiple digestion sites will be displayed. After that, compare the list of digestion sites on the E1U region with the MCS on the pGL3-Basic vector in an excel sheet. Subsequently, prepare forward and reverse primers for PCR that contain restriction endonucleae sites using commercial gene primer synthesis services or primer selection software.Select a forward primer located about two kilobases upstream of the E1U sequence and a reverse primer within the E1U sequence. Then, amplify the E1U genomic region using these primers with PCR 01 to one microgram of Bak and PCR Master Mix containing high fidelity taq polymerase. Afterward, verify the length of the amplified PCR product by comparing it against a one kilobase DNA ladder using 0.8%TAE agarose gel electrophoresis.Afterward, digest the obtained PCR product and the pGL3-Basic vector using restriction endonucleases specific for restriction digestion sites introduced into the forward and reverse primers. Then, verify the digestion of the PCR product using agarose gel electrophoresis. Cut the digested vector DNA and the two kilobase PCR band from the agarose gel.Next, extract the digested vector DNA and the two kilobase PCR band from the gel slices using a commercial SV gel and PCR clean-up systems kit. Measure the concentration of the purified PCR product and the purified vector using UV spectrometry. Subsequently, prepare the E1U reporter construct by ligating the purified restriction enzyme digested PCR product and the pGL3-Basic firefly luciferase reporter vector using the T4 DNA ligase quick ligation kit thereby producing the E1U reporter construct named pGL3 E1U.In this step, culture the TM4 cells in 24-well plates at an initial density of 200, 000 cells per well. Six hours after placing the cells in culture, transfect the cells with 0.2 micrograms of MP pGL3-Basic vector or 0.2 micrograms of pGL3 vector along with four nanograms of pRL-TK as an internal control. Thirty hours following transfection, remove the growth media from the transfected cells.Wash the cells with PBS, then remove the wash solution. After that, measure the firefly and Renilla luciferase activity using a commercial kit. Express the activity for each tube as the ratio of the firefly luciferase activity divided by the internal control.This step confirms the functional activity of the testis specific promoter. Here is the reporter assay for Bcrp1 promoters E1U, E1A, E1B, and E1C in bulbs these are totally cell-derived TM4 cells. Data are expressed as the luminescence of firefly luciferase normalized to that of Renilla luciferase.The asterisk indicates a significant difference from the MP pGL3 vector control. This graph shows the effects of mutation of the SF-1 response element in the Bcrp1 E1U reporter construct on luciferase activity. Bcrp1 reporter constructs with a putative SF-1 binding region were transfected into TM4 cells with and without the enforced expression of SF-1.In the figure, a denotes a significant difference from the empty vector pGL3 control;b signifies a statistically significant difference compared to the E1U promoter construct in the vector transfected cells. While attempting this procedure, it's important to remember to select an appropriate cell line for the reporter assay. The cell line should express the gene of interest under the control of the promoter in the reporter construct.Following this procedure, other methods, such as quantitative RT-PCR can be performed in order to facilitate evaluation of alternative promoter usage in multiple tissue samples. After watching this video, you should have a good understanding of how to predict alternative promoter usage for a gene of interest and to determine the functionality of that promoter.

Research

JoVE Journal

Biology

Preparation of 2-dGuo-Treated Thymus Organ Cultures

2 dGuo治疗胸腺器官培养的制备

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

科研

生物学

Dissection and Staining of Drosophila Larval Ovaries

Dissection and Staining of Drosophila Larval Ovaries

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

定量检测，以研究蛋白质：DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

基于荧光引物延伸法确定转录起点和核糖核酸酶的切割位点<em&gt;在体内</em

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

质粒载体产生表达FLAG标记的蛋白质在人延伸因子1α启动子的使用吉布森大会规则

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

将细胞质Foci分类为哺乳动物应激颗粒的方法

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

植物启动子分析: 菜豆根瘤特异启动子的鉴定与表征

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...