方法发现小鼠BCRP1的另类用法子和转录调控
1Greenebaum Cancer Center,University of Maryland School of Medicine, 2Pharmaceutical Sciences,University of Maryland School of Pharmacy, 3Baltimore VA Medical Center, 4Membrane Transport and Biopharmaceutics, School of Pharmaceutical Sciences,Kanazawa University, 5Obstetrics, Gynecology and Reproductive Science,University of Pittsburgh, 6Magee Women’s Research Institute, 7Obstetrics, Gynecology, Perinatal Research Branch (NICHD),Wayne State University School of Medicine, 8Medicine,University of Maryland School of Medicine, 9Pathology,University of Maryland School of Medicine, 10Pharmacology,University of Maryland School of Medicine, 11Experimental Therapeutics,University of Maryland School of Medicine
Summary
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。
Abstract
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。
Introduction
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> 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'cDNA末端（5'-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> 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。
Protocol
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'末端的小鼠BCRP1基因英寸通过输入mRNA的参考序列ID（NM_011920.3）进入ENSEMBL基因组浏…
Representative Results
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'外显子2的端部1 mRNA 的示于表1，与它们在基因组的位置?…
Discussion
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'-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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| COMMERCIAL BIOLOGIC MATERIALS AND KITS: | |||
| First Choice RLM-RACE kit | Ambion Inc., Austin, TX, currently available through Life Technologies | AM1700 | 5'-RACE PCR kit |
| TOPO TA cloning kit | Life Technologies | K450001 | This kit contains the TOP10 chemically competent E. coli bacteria. |
| T4 DNA ligase quick ligation kit | New England Biolabs | M2200 | |
| Faststart high-fidelity Taq- DNA polymerase | Sigma-Aldrich/Roche | 3553400001/RMB-4738284001 | Contains a high-fidelity Taq polymerase enzyme blend |
| XtremeGENE HP DNA transfection reagent | Roche | 6366236001 | |
| Dual-luciferase reporter assay kit | Promega | E1910 | Includes the pGL3-basic empty reporter vector (firefly luciferase), pRLTK renilla luciferase expressing vector, and other control vectors. |
| QIAprep | Qiagen | 27104 | For plasmid miniprep – isolation and purification of plasmid DNA from bacterial colonies |
| pCR2.1 vector | part of the TOPO TA cloning kit | ||
| pGL3-basic luciferase containing vector | Promega | E1751 | |
| pRL-TK Renilla luciferase expressing vector | Promega | E2241 | |
| Bacterial artificial chromosome | BACPAC Resources Center, Children’s Hospital Oakland Research Institute, Oakland, CA | RP23-285A12 and RP24-314E24 | http://bacpac.chori.org/clones.htm |
| Name | Company | Catalog Number | Comments |
| REAGENTS/CHEMICALS/MEDIA/SUPPLIES | |||
| TRIzol | Life Technologies | 15596026 | |
| Wizard® SV Gel and PCR Clean-Up System | Promega | A9281 | Useful for purifying PCR products following digestion with restriction endonucleases |
| CRYOVIALS | Denville Scientific | V9012 | |
| Name | Company | Catalog Number | Comments |
| SOFTWARE: | |||
| Ensembl software | Ensembl project | http://Ensembl.org | The Ensembl project produces genome databases for vertebrates and other eukaryotic species, and makes this information freely available online. |
| Blast software | NCBI | http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD= Web&PAGE_TYPE=BlastHome |
|
| Primer 3 software | Simgene | http://simgene.com/Primer3 | Useful for designing primers for 5'-RACE PCR |
| NCBI Nucleotide database | NCBI | http://www.ncbi.nlm.nih.gov/nucleotide/ | |
| Name | Company | Catalog Number | Comments |
| CELL LINES: | |||
| TM4 murine Sertoli cells | ATCC, Manassas, Virginia | CRL-1715™ | |
| Name | Company | Catalog Number | Comments |
| INSTRUMENTS: | |||
| Luminometer | Turner Biosystems | TD-20/20 | This is a relatively inexpensive, manually operated luminometer. Automated systems are also available from the same manufacturer that utilize 96 well plates. |
| NanoDrop spectrophotometers | Thermo Scientific, Inc. | NanoDrop 2000 | Spectrophotometer can use very small quantities of sample |
| DU 800 UV/VIS spectrophotometer and Nucleic Acid Analysis II software | BeckmanCoulter Inc, Fullerton, CA | DU 800 |
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Cite This Article
Natarajan, K., Xie, Y., Nakanishi, T., Moreci, R. S., Jeyasuria, P., Hussain, A., Ross, D. D. Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1. J. Vis. Exp. (111), e53827, doi:10.3791/53827 (2016).