pgl3-basic vector中文
PGL3-Basic Vector
Storage Conditions: Store the pGL3 Luciferase Reporter Vectors at –20°C and the glycerol stock of JM109 cells at –70°C. III. pGL3 Vector Maps and Sequence Reference Points The listings of restriction sites for the pGL3 Luciferase Reporter Vectors are provided in Section VIII.G–J. Note: The specific transcriptional characteristics of the pGL3 Vectors will vary for different cell types. This may be particularly true for COS cells, which contain the SV40 large T antigen. The SV40 large T antigen promotes replication from the SV40 origin, which is found in the promoter of the pGL3-Promoter and pGL3-Control Vectors. The combination of large T antigen and SV40 origin will result in a higher copy number of these vectors in COS cells, which in turn may result in increased expression of the reporter gene compared to other cell and vector combinations.
人NRAGE基因启动子转录活性的初步研究
人NRAGE基因启动子转录活性的初步研究吴寅子;张丽;温传俊【摘要】利用生物信息学软件搜寻NRAGE启动子区域的转录因子结合位点,并根据这些位点利用PCR技术克隆NRAGE启动子不同区域的缺失突变体,分别插入Luciferase报告载体pG13-Basic载体中,构成8种缺失突变体的报告基因表达载体.通过双荧光素酶体系检测在HEK-293细胞中NRAGE启动子不同区域的转录活性,从而确定起主要作用的启动子区域.结果发现NRAGE基因转录起始位点上游一300bp到-100 bp的区域起到主要的转录调控作用,%We analyzed human NRAGE promoter region by bioinformatic software and found putative binding sites of transcription factors, then 8 different fragments were amplified by PCR and the mutant promoters were inserted to upstream of the luciferase gene in luciferase report vector pGL3-Basic and detected the transcription activity by dual-lueifer-ase reporter assay systerm. At transcription level we detected that transcription enhancer elements may exist in the region -300bp to -100bp.【期刊名称】《南京师大学报（自然科学版）》【年(卷),期】2011(034)003【总页数】5页(P90-94)【关键词】NRAGE;双荧光素酶法;缺失突变;转录活性【作者】吴寅子;张丽;温传俊【作者单位】南京师范大学生命科学学院分子细胞生物学研究所,江苏,南京,210046;南京师范大学生命科学学院分子细胞生物学研究所,江苏,南京,210046;南京师范大学生命科学学院分子细胞生物学研究所,江苏,南京,210046【正文语种】中文【中图分类】Q78NRAGE(Neurotrophin receptor-interacting MAGE homologue，MAGE:melanoma antigen gene)基因是由Salhe在2000年通过酵母双杂交的方法筛选到的与p75NTR相互作用的蛋白．隶属于恶性黑色素瘤抗原家族成员，又称之为 MAGE-Dl［1］．定位于染色体Xp11．21－11.23上，含有13个外显子和覆盖第二到第十二个外显子的一个开放性阅读框(ORF)．编码778个氨基酸，构成分子量为86 kDa的蛋白［2］．NRAGE的保守性强，在大鼠和小鼠体内都有该基因的类似物，NRAGE在发育早期以及成年个体中各个组织中都有表达［3］，这就意味着NRAGE可能发挥着重要的作用．在过去的研究中也确实发现NRAGE在生长发育、凋亡、分化以及肿瘤的转移过程中都有着调节作用［4-13］．目前关于NRAGE在疾病发生发展中的作用研究较多，而对于基因启动子及转录活性调控方面的研究尚未见相关报道．本研究利用生物信息学技术预测出NRAGE基因5'端上游序列中的潜在启动子序列，并分析NRAGE启动子区域发现存在 GATA1，SRY，HIF1-α，SP1，MZF1等多种转录因子结合位点．我们在试验中以NRAGE转录起始位点上游740 bp至下游60 bp为研究对象，分别构建了8个启动子缺失突变体的报告基因质粒，并检测转录活性，发现－300 bp到－100 bp之间的序列对NRAGE的转录活性影响最大，这为进一步研究NRAGE表达调控机制提供了基本的实验依据．HEK-293细胞株、pGL3-Basic载体、内参照质粒pRL-TK为本实验室保存;小提质粒试剂盒(Bio-mega)、割胶回收试剂盒(Promega);DpnⅠ去甲基化酶(NEB)，PCR试剂、DNA marker、T4 DNA连接酶、限制性内切酶KpnⅠ、Xh oⅠ购自大连宝生物TAKARA公司;胎牛血清、DMEM购自Gibco公司;双荧光素酶报告基因检测试剂盒购自Promega公司;荧光化学发光微孔板检测仪购自Thermo公司．应用在线软件 Promoter Proscan Version1.7(http://www．thr．crt．nih．gov/molbio/proscan/)分析NRAGE转录起始位点上游1 500 bp，预测基本启动子区域，利用TFSEARCH(http://www．cbrc．jp/research/db/TFSEARCH．html)软件分析启动子区域的转录因子结合位点．根据生物信息学软件分析的NRAGE 基因启动子区域及转录因子结合位点，针对NRAGE基因转录起始位点上游的740 bp至下游的60 bp，利用primer 5.0设计8对引物，在上游引物5'端添加KpnⅠ酶切位点，在下游引物5'端添加XhoⅠ结合位点，引物序列如下:上游引物下游引物4个上游引物分别和2个下游引物配对扩增，合成8条启动子缺失突变序列，PCR 扩增程序为:94℃变性5 min，94℃ 30 s，52℃ 30 s，72℃ 30 s，30 个循环，72℃延伸10 min．1%琼脂糖凝胶电泳检测，PCR 产物经过纯化后用KpnⅠ，XhoⅠ双酶切8h，割胶回收后，用T4 DNA连接酶将片段与pGL3-Basic载体连接，16℃过夜．取5 μL转化DH5α感受态．挑取克隆提质粒，用KpnⅠ，XhoⅠ双酶切进行鉴定，最后测序鉴定阳性克隆．HEK-293细胞培养于含有10%胎牛血清的DMEM中，培养基中含有100 U/mL青霉素，100 U/mL链霉素，置于5%CO2，37℃恒温培养，48 h传代一次．将HEK-293细胞接种于24孔板中，使细胞在接种第二天生长到30% ～50%融合，将重组报告基因质粒以及pGL3-Basic分别和内参照海肾荧光素酶报告基因质粒pRL-TK通过磷酸钙转染法共转入细胞，每孔转染重组报告基因质粒500 ng，pRL-TK 50 ng．转染后8 h换液，48 h进行荧光素酶活性检测．转染48 h后收获细胞，弃去培养细胞的培养液，PBS洗细胞2～3次，完全吸净PBS，使用双荧光素酶报告基因检测试剂盒进行细胞裂解及荧光素酶活性检测实验．每孔中加入100 μL 1×PLB，放置在摇床上室温摇15 min后将裂解液吸入EP管，12 000r/min离心5 min，吸取上清进行检测．在荧光化学发光微孔板检测仪中放入专用96孔板，选择检测区域，每孔加入100 μL双荧光素酶报告基因试剂盒中的Luciferase Assay BufferⅡ及20 μL细胞裂解液，混匀并进行检测，得出萤火虫荧光素酶活性．再加入100 μL Stop＆Glo Buffer，混匀后检测得到海肾荧光素酶活性．各孔细胞的转染效率用海参荧光素酶活性校正，所得数值为萤火虫荧光素酶活性与海肾荧光素酶活性的比值．实验重复3次，数据采用SPSS11.0统计软件分析，计算标准差．组间差异采用t-检验进行比较．Promoter Proscan Version 1.7分析NRAGE基因5'端非编码区，结果表明转录起始位点上游379－1 30 bp之间为基本启动子区域．利用TFSEARCH分析转录调控区域的潜在转录因子结合位点，发现含有GATA1，SRY，HIF1-α，SP1，MZF1 等多种转录因子结合位点．(图1)分别将4条上游引物与2条下游引物配对进行PCR，扩增出8条NRAGE转录调控区域的序列缺失片段，分别命名为pF1-R1(－740～ +60)，pF2-R1(－510～+60)，pF3-R1(－300～ +60)，pF4-R1(－100～+60)，pF1-R2(－740～ +4)，pF2-R2(－510～ +4)，pF3-R2(－300～ +4)，pF4-R2(－100～ +4)．PCR 产物经纯化后利用KpnⅠ和XhoⅠ双酶切，酶切片段连接到同样用KpnⅠ和XhoⅠ双酶切的pGL3-Basic载体上，构建成荧光素酶表达质粒．挑选阳性克隆并通过KpnⅠ和XhoⅠ双酶切鉴定是否有释放片段，并测序鉴定阳性克隆，证明构建成功(图2)．以海肾荧光素酶表达载体pRL-TK为内参照，以pGL3-Basic为阴性对照，将克隆得到的8个NRAGE转录调控区域缺失突变体报告基因质粒转入HEK-293细胞中，48 h后检测荧光素酶表达活性，所得结果如图3．试验结果表明，与pGL3-Basic相比，插入NRAGE启动子区片段的报告基因质粒的转录活性均有不同程度的增加，其中pGL3-NRG-pF1-R1的转录活性最强，而缺失了－300到－100之间的序列后，转录活性明显下降，可见－300到－100之间可能存在起主要调控作用的转录增强子元件，这与软件分析的结果相吻合．缺失－740到－510之间的序列后转录活性稍有下降，但差异不大，－510到－300之间的序列基本对转录活性没有影响，R2组与R1组相比分别缺失的是转录起始位点后4到60之间的序列，转录活性变化不大，可见这些区域并不是主要调控区域．本实验选用HEK-293细胞，因其具有永生化、转染效率高、培养条件低、增殖周期短等诸多优点，初步证明了NRAGE基因启动子－300到－100区域起到主要的转录调控作用．NRAGE基因在细胞存活、凋亡以及细胞周期和分化，肿瘤的发生和转移中都发挥着重要调节功能．而且在很多肿瘤细胞中，NRAGE的蛋白表达量都会发生异常［14］，这些可能与NRAGE基因的转录调控有关，然而目前国内外对于NRAGE 的研究多在其功能方面，对于NRAGE的具体转录调控机制还没有涉及．了解NRAGE基因的启动子区域是研究其表达调控的前提．要进行NRAGE启动子活性的研究，首先必须对上游调控序列进行克隆．本实验通过构建含不同长度NRAGE启动子片段的荧光素酶报告基因缺失质粒，瞬时转染细胞，检测荧光素酶相对活性，确定NRAGE基因启动子所在区域及主要调控区域．结果表明:与空载体相比，pGL3-NRG-pF1-R1的启动子活性最强，说明NRAGE的核心启动子存在于－740到60之间，而通过荧光素酶相对活性分析显示，－300到－100之间可能存在转录增强子元件，缺失此区域后，NRAGE启动子的转录活性明显降低．通过软件分析此区域的转录因子结合位点发现，－300到－100之间含有一个ADR1和一个SP1结合位点．下一步的工作将主要针对这2个转录因子，确定具体是何转录因子在NRAGE的转录过程中起到主要调控作用．综上所述，本研究初步探索了NRAGE基因5'端上游序列不同区域在HEK293细胞中转录活性的差异，确定了NRAGE基因的核心启动子所在区域，对于研究NRAGE基因转录调控元件及阐明该基因的表达调控机制奠定了基础．进一步分析与NRAGE转录调控相关的转录因子将有利于深入探讨癌症的发生与转移机理，有利于寻找更好的干预治疗靶点．【相关文献】［1］ Imai Y，Shichijo S，Yamada A，et al．Sequence analysis of the MAGE gene family encoding human tumor-rejection antigens［J］．Gene，1995，60(2):287-290．［2］ Zhang C G，Xin G C，Wei H D，et al．A new melanoma antigen-encoding gene subfamily in human chromosome X［J］．Acta Genetica Sinica，2001，28(3):197-203．［3］ Kendall S E，Goldhawk D E，Kubu C，et al．Expression analysis of a novel p75NTR signaling protein，which regulates cell cycle progression and apoptosis［J］．Mech Dev，2002，117(1/2):187-200．［4］ Salehi A H，Roux P P，Kubu C J，et al．NRAGE，a novel MAGE protein，interacts with the p75 neurotrophin receptor and facilitates nerve growth factor dependent apoptosis［J］．Neuron，2000，27(2):279-288．［5］ Wen C J，Xue B，Qin W X，et al．hNRAGE，A human neurotrophin receptor interacting MAGE homologue，regulates p53 transcriptional activity and inhibits cell proliferation［J］．FEBS Lett，2004，564(1/2):171-176．［6］ Salehi A H，Xanthoudakis S，Barker P A．NRAGE，a p75 neurotrophin receptor interacting protein，induces caspase activation and cell death through a JNK dependent mitochondrial pathway［J］．J Biol Chem，2002，277(50):48 043-48 050．［7］ Williams M E，Strickland P，Watanabe K，et al．UNC5H1 induces apoptosis via its Juxtamembrane region through an interaction with NRAGE［J］．Biol Chem，2002，278(19):17 483-17 490．［8］ Masuda Y，Sasaki A，Shibuya H，et al．Dlxin-1，a novel protein that binds Dlx5 and regulates its transcriptional function［J］．Biol Chem，2001，276(7):5 331-5 338．［9］ Bertrand M J M，Kenchappa R S，Andrieu D，et al．NRAGE，a p75NTR adaptor protein，is required for developmental apoptosis in vivo［J］．Cell Death Differ，2008，15(12):1 921-1 929．［10］ Matluk N，Rochira J A，Karaczyn A，et al．A role for NRAGE in NF-kappaB activation through the non-canonical BMP pathway［J］．BMC Biology，2010，8:7-16．［11］ Chu C S，Xue B，Tu C，et al．NRAGE suppresses metastasis of melanoma and pancreatic cancer in vitro and in vivo［J］．Cancer Lett，2007，250(2):268-275．［12］ Du Q，Zang Y，Tian X X，et al．MAGE-D1 inhibits proliferation，migration and invasion of human breast cancer cells［J］．Oncol Rep，2009，22(3):659-665．［13］ Xue B，Wen C J，Shi Y，et al．Human NRAGE disrupts E-cadherin/b-catenin regulated homotypic cell-cell adhesion，Biochem．Biophys［J］．Res Commun，2005，336(1):247-251［14］张成岗，贺福初．黑色素瘤抗原编码基因家族新成员MAGED1的组织表达谱研究［J］．中国生物化学与分子生物学报，2002，18(2):165-171．。
同型半胱氨酸对FABP4启动子活性的影响
第28卷 第29期 中国现代医学杂志 Vol. 28 No.29 2018年10月 China Journal of Modern Medicine Oct. 2018收稿日期：2018-05-15*基金项目：国家自然科学基金（No ：81560084）；宁夏自然科学基金（No ：NZ16063）；宁夏高等学校科学研究项目（No ：NGY2016087）[通信作者] 姜怡邓，E-mail ：nyxxgjyd@DOI: 10.3969/j.issn.1005-8982.2018.29.005文章编号： 1005-8982（2018）29-0025-06同型半胱氨酸对FABP 4启动子活性的影响*熊建团1，李旭生2，杨安宁3，杨松昊3，邓梅3，王磊3，高源3，李南1，杨晓玲3，贾月霞3，姜怡邓3（1.宁夏医科大学 药学院，宁夏 银川 750004；2.宁夏医科大学总医院 骨科，宁夏 银川750004；3.宁夏医科大学 基础医学院，宁夏 银川 750004）摘要：目的 通过克隆脂肪酸结合蛋白4（FABP4）基因启动子，确定其活性核心区和功能片段，分析心血管疾病独立危险因子同型半胱氨酸（Hcy）对FABP 4启动子活性的影响。
方法 应用生物信息学预测FABP 4基因启动子区顺式转录作用元件和反式作用因子，以pGL3-Basic 为载体，采用基因重组法构建启动子截取片段，转染HEK-293A 细胞，观察不同截取片段的荧光素酶活性变化，确定活性最强的片段。
进一步将核心启动子片段（-2000/-1）转染巨噬细胞，观察不同浓度Hcy 和DNA 甲基化抑制剂5-氮杂胞苷（AZC）对FABP 4基因启动子活性的影响。
结果 不同长度截取片段转染HEK-293A 细胞后检测荧光素酶活性结果显示，与pGL3对照组比较，-2000/-1片段转录活性最强。
将核心启动子片段（-2000/-1）转染巨噬细胞并用不同浓度Hcy 干预后，与0μmol/L Hcy 组比较，100和200μmol/L Hcy 组启动活性升高；与100μmol/L Hcy 组比较，在100μmol/L Hcy 基础上用AZC 干预后，FABP 4基因启动活性升高（P <0.05）。
AP1与NF-κB对PPARδ基因的转录调控作用
AP1与NF-κB对PPARδ基因的转录调控作用蒋晓刚;韩燕;杨旭东;毛泽斌【摘要】目的研究在过氧化物酶体增长因子活化受体δ(PPARδ)表达中的相关调控因子.方法用RT-PCR扩增人类PPARδ启动子序列,通过Deletion及观测各重组体转染活性,通过电泳迁移率变更分析(EMSA)、突变等方法确定核转录因子激活蛋白1(APl)及NF-κB对PPARδ表达的作用.结果 Deletion及重组转染后发现AP1及NF-κB因子各自结合位点突变后转染活性明显降低,同时突变其转染活性无明显变化.结论 AP1及NF-κB均可以增强PPARδ的表达活性.%Objective To study the regulatory effects of activator protein 1 (AP1) and NF-KB on the PPARδ gene in Lovo cells. Methods By using methods of deletion, transient transfection, Gel-Shift and mutation, the regulatory effects of AP1 and NF-ΚB on PPARδ gene in Lovo cells were observed. Results Transient transfection results showed that the regions from 3 361 to 3 464 were sufficient to confer increased expression in Lovo cells. And there was the region involved in protein binding as shown in Gel-Shift assays. Mutation suggested that the AP1 and NF-ΚB within the 5'-UTR of PPARδ constituted a binding site for a potential transcriptional activator in Lovo cells. Conclusion Our data indicate that AP1 and NF-ΚB are potential transcriptional enhancers for PPARδ.【期刊名称】《西安交通大学学报（医学版）》【年(卷),期】2012(033)005【总页数】4页(P560-563)【关键词】过氧化物酶体增长因子活化受体δ;核转录因子激活蛋白-1;NF-κB【作者】蒋晓刚;韩燕;杨旭东;毛泽斌【作者单位】西安交通大学医学院遗传与分子生物学系,陕西西安710061;西安交通大学医学院遗传与分子生物学系,陕西西安710061;西安交通大学医学院遗传与分子生物学系,陕西西安710061;北京大学医学部生化系,北京853006【正文语种】中文【中图分类】R392.11过氧化物酶体增长因子活化受体（peroxisome proliferator activated receptor，PPAR）属于核激素受体超家族的新成员［1］，是由英国科学家ISSEMAN和GREEN于1990年首先发现［2］。
报告基因和荧光淬灭
利用某种物质对某一种荧光物质的荧光猝灭作用而建立的对该猝灭剂 的荧光测定方法，即为荧光猝灭法
荧光猝灭法比直接荧光测定法更为灵敏，具有更高的选择性
肿瘤转移过程
应用举例：
MMP-2荧光分析药物筛选原理图
加 入 底 物 Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 ， 在 37C温育过程中，MMP-2水解底物，释放出Mca发光基团和 Dpa淬灭基团。此时可以检测到荧光，随着时间延长，两种基团 浓度增加，基团之间相互碰撞机率增大，导致发光基团荧光逐渐 被淬灭。
荧光显微镜
多管水母科：Aequorleidae 27-30kD, 395nM
激光共聚焦显微镜
海紫罗兰科：Renillidae 54 kD, 498nM
荧光激活的细胞分拣器（FACS）
共定位
FRET，荧光能量共振转移
FRET，即荧光能量共振转移(fluorescence resonance energy transfer,) 随着绿色荧光蛋白应用技术的发展，成为检测活体中生物大分子纳米级距离和 纳米级距离变化的有力工具，在生物大分子相互作用分析、细胞生理研究、免 疫分析等方面有着广泛的应用。 原理：荧光能量共振转移是距离很近的两个荧光分子间产生的 一种能量转移现 象。当供体荧光分子的发射光谱与受体荧光分子的吸收光谱重叠，并且两个分 子的距离在10nm范围以内时，就会发生一种非放射性的能量转移，即FRET现象， 使得供体的荧光强度比它单独存在时要低的多（荧光猝灭），而受体发射的荧 光却大大增强（敏化荧光）。
发光机理：
BL 以脂肪醛（RCHO）为底物，在还原型黄素单核苷酸（FMNH2）及氧 脂肪醛氧化成脂肪酸+光子（490n荧光）
钠碘同向转运体相关文献4
ORIGINAL ARTICLETargeting of tumor radioiodine therapy by expression of the sodium iodide symporter under control of the survivin promoterR Huang 1,2,4,Z Zhao 1,2,4,X Ma 1,2,S Li 1,2,R Gong 3and A Kuang 11Department of Nuclear Medicine,National Key Discipline of Medical Imaging and Nuclear Medicine,West China Hospital,Sichuan University,Chengdu,China;2State Key Laboratory of Biotherapy,West China Hospital,Sichuan University,Chengdu,China and 3Department of Thyroid Surgery,West China Hospital,Sichuan University,Chengdu,ChinaTo test the feasibility of using the survivin promoter to induce specific expression of sodium/iodide symporter (NIS)in cancer cell lines and tumors for targeted use of radionuclide therapy,a recombinant adenovirus,Ad-SUR-NIS,that expressed the NIS gene under control of the survivin promoter was constructed.Ad-SUR-NIS mediating iodide uptake and cytotoxicity was performed in vitro .Scintigraphic,biodistribution and radioiodine therapy studies were performed in vivo .PC-3(prostate);HepG2(hepatoma)and A375(melanoma)cancer cells all exhibited perchlorate-sensitive iodide uptake after infection with Ad-SUR-NIS,B 50times higher than that of negative control Ad-CMV-GFP-infected cells.No significant iodide uptake was observed in normal human dental pulp fibroblast (DPF)cells after infection with Ad-SUR-NIS.Clonogenic assays demonstrated that Ad-SUR-NIS-infected cancer cells were selectively killed by exposure to 131I.Ad-SUR-NIS-infected tumors show significant radioiodine accumulation (13.3±2.85%ID per g at 2h post-injection),and the effective half-life was 3.1h.Moreover,infection with Ad-SUR-NIS in combination with 131I suppressed tumor growth.These results indicate that expression of NIS under control of the survivin promoter can likely be used to achieve cancer-specific expression of NIS in many types of cancers.In combination with radioiodine therapy,this strategy is a possible method of cancer gene therapy.Cancer Gene Therapy (2011)18,144–152;doi:10.1038/cgt.2010.66;published online 29October 2010Keywords:sodium iodide symporter;survivin promoter;radioiodine therapyIntroductionThe sodium/iodide symporter (NIS),an intrinsic mem-brane glycoprotein with 13putative transmembrane domains,has an important role in the biosynthesis of thyroid hormones by mediating the active transport of iodide into thyrocytes.1–3NIS is critical for the diagnosis and therapeutic management of thyroid diseases,includ-ing thyroid carcinoma.Cloning of the rat and human NIS genes in 1996and extensive characterization of these genes have paved the way to novel radionuclide gene therapy strategies.1,4Through targeted transfer and expression of the NIS gene,radioiodine treatment could be used to treat non-thyroid malignant disease in addition to thyroid carcinoma.Tumor-specific expression of the NIS gene has been performed with promising results in a variety of tumors,including prostate carcinoma,5–9hepatocellular carci-noma,9–12medullary thyroid carcinoma,13colon cancer,14breast cancer 15and neuroendocrine tumors.16For exam-ple,prostate tumors were successfully treated in vivo with systemic administration of 131I,188Re and 211At following selective expression of NIS under control of the prostate specific antigen promoter.Although previous studies have been promising,the use of such tissue-specific promoters has a limited scope.Therefore,the identification of a more general tumor-specific promoter to regulate NIS expression could be useful for expanding this strategy for treatment of a wide variety of tumors.Survivin,a member of the inhibitor of apoptosis gene family,is usually expressed in the embryonic lung and fetal organs in the developmental stages but undetectable in normal adult tissues other than the thymus,placenta,CD34þstem cells and basal colonic epithelial cells.17–21However,survivin seems to be selectively expressed in transformed cells and in most human cancers,including lung,breast,pancreatic and colon carcinomas,soft tissue sarcomas,brain tumors,melanoma,neuroblastoma and hematologic malignancies.22Clinical studies have indicated a positive correlation between high survivinReceived 7February 2010;revised 22June 2010;accepted 2September 2010;published online 29October 2010Correspondence:Professor A Kuang,Department of Nuclear Medicine,National Key Discipline of Medical Imaging and Nuclear Medicine,West China Hospital,Sichuan University,Chengdu 610041,China.E-mail:kuanganren@ 4These two authors contributed equally to this work.Cancer Gene Therapy (2011)18,144–152r2011Nature America,Inc.All rights reserved 0929-1903/11 /cgtexpression levels and poor prognosis,accelerated rate of recurrence and increased resistance to therapy in many cancer patients.23Previous studies have identified a putative region of the survivin promoter that is likely responsible for the induction of specific,high-level expression in tumors.24,25The survivin promoter contains multiple cell cycle-dependent elements and a cell cycle gene homology region.This region has been suggested to control expression of various G2-M-regulated genes, including the survivin gene,in a manner correlating with G2-M cell cycle periodicity.24In this study,we have developed a tumor-targeting adenovirus that expresses the NIS gene under control of the survivin promoter.We investigated the utility of this system for molecular imaging and radioiodine gene therapy.The results reported herein demonstrate the potential efficacy and selectivity of a combination of radioiodine therapy and adenovirus-mediated expression of the NIS gene under the control of the survivin promoter for cancer therapy.Materials and methodsCell linesThe human prostate cancer cell line,PC-3;the human hepatocellular carcinoma cell line,HepG2;the human melanoma cell line,A375and the adenoviral transformed human embryonic kidney cell line,293,were purchased from the American Type Culture Collection(ATCC, Rockville,MD).All cancer cell lines were cultured in RPMI1640medium supplemented with10%calf serum (Gibco,Carlsbad,CA),2m M glutamine,100IU ml–1 penicillin and100ng ml–1streptomycin.Cells were main-tained at371C in a humidified atmosphere containing5% CO2.Both293cells and a control human dental pulp fibroblast cell line(DPF;a kind gift from Dr Peng Li, West China College of Stomatology,Sichuan University) were cultured in Dulbecco’s Modified Eagle Medium (high glucose)medium supplemented with10%FBS and 100IU ml–1penicillin/streptomycin(100ng ml–1).Construction of plasmids and recombinant adenovirus In order to generate the luciferase expression plasmid under the control of the survivin promoter(pSUR-LUC), an B260bp fragment of the human survivin gene promoter(B222toþ39,GeneBank Accession Number AY795969)was amplified from PC-3genomic DNA by PCR.This region contains two cell cycle-dependent elements and one cell cycle gene homology region.The sequences of the oligonucleotide primers were as follows: forward primer,50-AGATCTGCACGCGTTCTTTGAA AGC-30,and0-AAGCTTCCACCTCTG CCAACGGGTC-3,which allowed for the introduction of Bgl II and Hin dIII sites(underlined in the respective sequences).The survivin promoter was T/A cloned into the PMD-T18vector for sequencing,and then subcloned using the Bgl II and Hin dIII sites into the luciferase vector pGL3-basic(Promega,Madison,WI)to generate pSUR-Luc(Figure1a).In order to generate the NIS constructs,the coding region of NIS was amplified from normal human thyroid tissue by RT–PCR(nucleotides348to2280,GeneBank Accession Number,NM_000453),followed by T/A cloning into the PMD-T18vector for sequencing.The sequences of the oligonucleotide primers were as follows: forward primer,50-CCCAAGCTTATGGAGGCCGTG GAGACCGGGGAAC-30,and reverse primer,50-GCG TCTAGATCAGAGGTTTGTCTCCTGCTG-30,which allowed for the introduction of Hin dIII and Xba Isites(underlined in the respective sequences).The luciferase gene of pSUR-Luc was replaced with hNIS by subcloning into the Hin dIII and Xba I sites.The survivin promoter-hNIS-SV40polyA cassette was then isolated by digestion with Kpn I and Sal I and cloned into the pShuttle-1vector(Stratagene,Cedar Creek,MD)to generate pS-SUR-NIS(Figure1b).Subsequently,homo-logous recombination was performed using the Adeasier system(Stratagene)in BJ5183Escherichia coli to generatepAd-SUR-NIS.The Ad-SUR-NIS virus was packaged by transfecting4m g of Pac I-digested pAd-SUR-NIS DNAinto293cells using Lipofectamine2000(Invitrogen, Carlsbad,CA)and propagating in293cells according tothe standard procedure.After two-step purification onCsCl gradients,viral stocks were desalted using Pharma-cia G50columns(Orsay,France)and frozen atÀ801C in10mM Tris-HCl(pH7.5)containing2.5%glycerol.Viraltiters were calculated by a dilution plaque assay in293cells and expressed as PFU per ml.Purification and plaque assays were performed by SinoGenoMax,Beijing, China.The recombinant adenovirus,Ad-CMV-NIS, which uses the CMV promoter to drive NIS expression (previously described26),was used as a positive control.The recombinant adenovirus,Ad-CMV-GFP,which provided by SinoGenoMax,was used as a negativecontrol.Figure1Structures of plasmids used in this study.(a)Diagram represents the structure of the luciferase reporter plasmid.The survivin promoter(À222to39bp)was cloned upstream of the luciferase gene using the restriction sites indicated.(b)Diagram represents the structure of the adenovirus shuttle plasmid pS-SUR-NIS.The plasmid was constructed by inserting a DNA fragment intothe E1-deleted region using the Kpn I/Sal I restriction sites.This DNA fragment contained the survivin promoter,the full length NIS cDNA,and the Simian Virus40polyadenylation signal.Targeted radioiodine therapy for tumorsR Huang et al145Cancer Gene TherapyAnalysis of the survivin promoter activity in vitroIn order to determine the transcriptional activation activity of the survivin promoter,we utilized the Dual-Luciferase reporter assay system(Promega).The plasmid pRL-TK,which contains the Renilla luciferase gene under the control of the thymidine kinase promoter, was used as an internal control.In all,1Â106cells of each cancer cell line and normal control were plated in six-well tissue culture plates and24h later,the cells were transfected with4m g of the indicated plasmid DNA and 0.1m g of pRL-TK using Lipofectamine2000(Invitrogen) according to the manufacturer’s protocol.At48h post-transfection,luciferase activity was determined using the Reporter Lysis Buffer and Luciferase Assay System according to the manufacturer’s protocol(Promega). The dual luciferase ratio was defined as the luciferase activity of the indicated plasmid divided by the luciferase activity of pRL-TK.The pSV40-LUC vector(Promega) that contains Simian Virus40(SV40)promoter was used as a positive control.All experiments were performed in triplicate.In vitro125I and99m Tc pertechnetate uptake experimentsIn all,1Â106cells of each cancer cell line and control DPF cells were plated in six-well tissue culture plates and allowed to reach exponential growth for24h and infected with Ad-SUR-NIS,Ad-CMV-NIS(positive control)or Ad-CMV-GFP(negative control)viruses at MOI¼100in 1ml of medium without serum.After2h,the media was changed to complete medium.For each cell line,the medium used for infection was the same as the culture medium.Approximately28–30h after viral infection,cells were washed once with1ml of phosphate-buffered saline (PBS).Iodide uptake was then initiated by adding1ml of medium without serum containing3.7KBq of125I or 99m Tc pertechnetate per well.Following30min incuba-tion with radionuclide,cells were washed twice with ice-cold PBS and incubated for20min in1ml of ice-cold ethanol.The ethanol was then recovered,and radio-activity was quantified(c.p.m.)using a g-counter(No.262 Factory,Xi’an,China).For inhibition experiments,cells were incubated with both300m M NaClO4and125I for 30min,followed by quantification of125I uptake as described above.In vitro cell killing with131I and clonogenic assayCell killing with131I was performed according to the method of Boland et al.27described as following:cells were seeded in24-well tissue culture plates and divided into three groups.The first two groups were infected with the Ad-SUR-NIS adenovirus and the Ad-CMV-GFP virus,respectively.Thirty hours after infection,cells were washed once with PBS and incubated with1ml of serum-free medium containing370KBq131I.The third group was not infected and was incubated in serum-free medium.After7h,cells from all three conditions were washed twice with PBS,trypsinized,and counted.For each condition,cells were plated in triplicate in six-well tissue culture plates(1000cells per well)and incubated for 1week at371C.Cells were then washed once with PBS and stained with crystal violet solution(2g l–1crystalviolet,4%formaldehyde,20%ethanol).Colonies of430cells were counted,and the mean and s.d.of the number of colonies were determined for each condition.Resultswere expressed as the percentage of surviving cells,defined as the percentage of colonies obtained after treatment with131I compared with no intervention,and are representative of two independent experiments. Biodistribution of125I in tumor-bearing mice Approximately2Â107PC-3cells were subcutaneouslytransplanted into the left or right flank of6-week-old Balb/c nude mice and allowed to proliferate until the tumor reached a diameter of8–10mm.At this point, the Ad-SUR-NIS virus or the Ad-CMV-GFP virus (negative control)was injected into the tumors(both 1Â109PFU).Radionuclide uptake was assessed2days later by intravenous injection with370KBq of125I.At2, 4,6and12h post-injection,animals were killed,and selected organs(bone,muscle,heart,lung,liver,spleen, stomach,intestine,kidney,brain and tumors)were dissected,weighed and counted for radioactivity. Scintigraphic imaging of tumor-bearing mice in vivo Forty-eight hours after intratumoral injection of 1Â109PFU Ad-SUR-NIS or Ad-CMV-GFP(negative control),PC-3tumor-bearing mice were injected with 18.5MBq of99m Tc pertechnetate via the tail vein.After 2h,mice were anesthetized with ketamine and xylazine and scanned with a g-camera equipped with a pinhole collimator(Philips Medical Systems,Milpitas,CA). Effects of NIS expression on131I therapy in vivo Accordingly,the PC-3tumors were induced in Balb/c nude mice as described above.When the tumors reached 8–10mm in diameter,the mice were given thyroxine (Sigma-Aldrich,St Louis,MO)at a concentration of 5mg l–1in the drinking water for7days before treatment. Two days before treatment,the animals were divided into two groups of three animals each.One group received an intratumoral injection of1Â109PFU Ad-SUR-NIS.The other group received an intratumoral injection of 1Â109PFU Ad-CMV-GFP.Forty-eight hours later, both groups were given an intraperitoneal dose of 111MBq of131I(the dose same with Schipper et al.16at their NIS mediate-131I therapy experiment).The length and width of the tumors were measured with a caliper at 5-day intervals after treatment.Tumor size was calculated using the formula:lengthÂwidth2/2.All mice were followed for a total of30days and then euthanized. Tumor samples and immunohistochemical stainingIn order to detect NIS expression,resected tumors from nude mice were fixed in4%paraformaldehyde for24h. Samples were immunostained using a standard streptavidin-biotin labeling protocol.Briefly,after deparaffinization and antigen retrieval,5m m tissue sections were incubated with NIS antibody(mouse monoclonal,1:100,Chemicon, Temecula,CA)at371C for1h then at41C overnight.Targeted radioiodine therapy for tumorsR Huang et al 146Cancer Gene TherapyThe sections were then incubated with biotinylated goat anti-mouse immunoglobulin G(1:200,Zymed Labora-tories,San Francisco,CA)and subsequently incubated with horseradish peroxidase-labeled streptavidin(1:200, Zymed Laboratories).The cells were then counterstained with3,30-diaminobenzidine for analysis.Statistical analysisAll data are expressed as mean±s.d.Significance was determined by the Student’s t-test.P o0.05was considered to be statistically significant.ResultsCancer-specific activation of the survivin promoterin vitroTo assess whether the activation of the survivin promoter is cancer specific and to compare the transcriptional activation activity between the survivin promoter and the SV40promoter,we transfected both cancer(PC-3, HepG2,A375)and normal(DPF)cells with plasmids containing the luciferase gene under control of either the survivin promoter(pSUR-Luc)or the SV40promgfeoter (pSV40-Luc).Survivin promoter activity was generally very high in cancer cell lines and very low in normal cells.The mean dual reporter ratio for cancer and normal cell lines was 6.76±0.83and0.33±0.23,respectively (Figure2).However,the activity of the SV40promoter was similar in both cancer cell lines and normal cells,with a mean dual reporter ratio of2.23±0.08and2.23±0.19, respectively(Figure2).These data suggest that the survivin promoter is selectively active in cancer cells compared with normal cells.Ad-SUR-NIS is functional in various human cancer cell linesAn adenovirus that expresses NIS under control of the survivin promoter(Ad-SUR-NIS)or the CMV promoter (Ad-CMV-NIS)and a control virus expressing GFP under control of the CMV promoter(Ad-CMV-GFP) were generated and used to infect cancer cell lines(PC-3, HepG2,A375)and non-malignant control cells(DPF).In order to test the expression of NIS in these cell lines, functional assays quantifying radioiodine uptake were performed.Thirty hours after adenovirus infection,iodide uptake was measured by incubating the cells for30min with3.7KBq of125I.As shown in Figure3,all cancer cell lines infected with Ad-SUR-NIS exhibited significant iodide uptake,with a mean value of12092±977c.p.m. This value represented a45–56-fold increase over control Ad-CMV-GFP-infected cells(237±36c.p.m.,P¼0.000). No significant iodide uptake was evident in DPF cells infected with Ad-SUR-NIS(300±42c.p.m.),consistent with luciferase reporter assays indicating the specificity of the survivin promoter for cancer cells(Figure3).Both cancer and normal cell lines infected with the general NIS expression virus Ad-CMV-NIS exhibited significant iodide uptake.The mean amount of iodide uptake for all Ad-CMV-NIS-infected cells was23256±3806c.p.m.,B85–120times higher than Ad-CMV-GFP-infected cells, indicating the efficacy of NIS expression for induction of radioiodine uptake(Figure3).In addition to125I uptake, cancer cell lines infected with Ad-SUR-NIS also exhibited selective and increased99m Tc pertechnetate uptake.The mean amount of99m Tc pertechnetate uptake was 11906±1424c.p.m.for cancer cells(Figure3).In order to further demonstrate that uptake of125I wasdependentFigure2In vitro analyses of transgene expression of the survivinand SV40promoters by transient transfection.The transcriptional activity of the survivin and SV40promoters was measured in various cancer and normal cells.The data shown are the means±s.d.of atleast three independentexperiments.Figure3In vitro radioiodine and99m Tc pertechnetate uptake.PC-3,HepG2and A375cancer cell lines and the control DPF cell linewere infected with Ad-SUR-NIS,Ad-CMV-NIS or Ad-CMV-GFP viruses and subjected to either125I or99m Tc pertechnetate uptake.Ad-SUR-NIS-infected PC-3,HepG2and A375cancer cell lines allshow significant125I and99m Tc pertechnetate uptake ability compared with Ad-CMV-GFP-infected controls.No significant uptakewas observed in Ad-SUR-NIS-infected DPF cells.Ad-CMV-NIS infection resulted in increased125I uptake in all cell lines.Cotreat-ment with NaClO4completely inhibited125I uptake in Ad-SUR-NIS-infected cancer cells.Targeted radioiodine therapy for tumorsR Huang et al147Cancer Gene Therapyupon NIS expression in Ad-SUR-NIS-infected cells,cells were treated with the specific NIS inhibitor,sodium perchlorate during iodide uptake.Figure3shows that sodium perchlorate treatment inhibited iodide accumula-tion by92.5±1.2%(P¼0.000).Ad-SUR-NIS-infected human cancer cells are efficiently killed by131I in vitroIn order to demonstrate selective killing of cancer cells that could be obtained using Ad-SUR-NIS infection in combination with radioactive iodide treatment,131I uptake experiments were performed on Ad-SUR-NIS-infected PC-3,A375,HepG2and DPF cells.Ad-CMV-GFP-infected cells treated with131I were used as controls. After131I treatment,clonogenic assays were performed, and results were expressed as the percentage of surviving cells compared with untreated controls.Following ex-posure to131I,o10%of Ad-CMV-GFP-infected cells were killed compared with untreated controls.However, 86±3.5%,92±3.0%and89±4.5%of PC-3,A375and HepG2cells,respectively,were killed by131I following infection with Ad-SUR-NIS.No significant cell death was observed in Ad-SUR-NIS-infected DPF cells treated with131I,as91±2.3%of cells were recovered.These results demonstrate that coupling Ad-SUR-NIS infection and131I treatment efficiently and specifically leads to cancer cell killing in vitro.125I biodistribution studies in tumor-bearing miceIn order to extend our in vitro results using the Ad-SUR-NIS virus to an in vivo model,we used nude mice bearing tumors generated by subcutaneous transplantation of PC-3cells as a model.Tumors were injected with either the Ad-SUR-NIS virus or the control Ad-CMV-GFP virus,and mice were subjected to125I treatment48h later. Quantitation of the uptake of125I(%ID per g)in tumors and various organs was evaluated at2,4,6and12h after 125I administration(Table1).PC-3tumors infected with Ad-SUR-NIS accumulated13.3±2.85%ID per g at2h, 5.61±1.38%ID per g at4h,2.74±0.21%ID per g at6h and0.68±0.05%ID per g at12h,with a mean effective half-life of 3.1±0.6h.All other organs accumulated negligible amounts of isotope(below2.5%ID per g at all times)except stomach.The biodistribution data are summarized in pared with Ad-SUR-NIS-infected tumors,negative control tumors infected with Ad-CMV-GFP accumulated0.66±0.24%ID per g125I at 2h after administration(P¼0.000).At4,6and12h, they accumulated0.42±0.44%ID per g(P¼0.001), 0.34±0.04%ID per g(P¼0.000)and0.06±0.01%ID per g(P¼0.014),respectively.These data indicate that infection of tumors with the Ad-SUR-NIS virus leads to selective uptake of125I in vivo.Visualization of Ad-SUR-NIS-infected tumors by99m Tc pertechnetate scintigraphyIn order to test whether Ad-SUR-NIS-infected tumors could be visualized in vivo,scintigraphy was performed on tumor-bearing mice using99m Tc pertechnetate,allowing imaging with a pinhole collimator.99m Tc pertechnetate (18.5MBq)was injected intraperitoneally,and images were taken after2h(Figure4).As expected,tissues naturally expressing NIS(stomach and thyroid)or involved in iodide elimination(bladder)were visualized. The Ad-SUR-NIS-infected tumor was clearly visible,with intensity comparable to that of the thyroid or the bladder. However,the Ad-CMV-GFP-infected PC-3tumor was not visible.Decreased tumor growth induced by Ad-SUR-NIS infection in combination with131I treatmentIn vitro studies suggested that Ad-SUR-NIS infection combined with radioiodine treatment can inhibit growth of cancer cells.In order to extend these results to an in vivo model,tumors were generated in Balb/c nude mice and infected with either Ad-SUR-NIS or Ad-CMV-GFP. After48h,mice were treated with131I as described in the Materials and methods.As shown in Figure5,tumors inTable1Biodistribution of125I pertechnetate in mice with Ad-SUR-NIS-injected PC-3xenografts(%ID per g tissue;mean and s.d.after intravenous injection)Organ2h4h6h12h Stomach14.7±0.729.42±1.59 4.44±0.860.54±0.12 Tumor13.3±2.85 5.61±1.38 2.74±0.210.68±0.05 Heart 1.25±0.230.31±0.270.29±0.100.14±0.01 Liver 1.21±0.260.45±0.250.46±0.170.20±0.04 Spleen 1.00±0.310.53±0.260.56±0.280.12±0.01 Lung 1.48±0.340.66±0.250.53±0.260.15±0.02 Kidney 2.10±0.510.89±0.190.66±0.270.16±0.03 Intestinal 1.78±0.690.37±0.430.53±0.290.17±0.03 Muscle0.60±0.160.40±0.090.37±0.010.07±0.02 Bone 1.44±0.330.84±0.090.48±0.080.28±0.01 Brain0.16±0.040.04±0.110.07±0.010.05±0.03Figure499m Tc pertechnetate scintigraphic images of PC-3 xenografts infected with either Ad-CMV-GFP or Ad-SUR-NIS.Mice harboring PC-3xenograft tumors infected with either Ad-CMV-GFP (a)or Ad-SUR-NIS(b)were imaged with a pinhole collimator2h after injection of18.5MBq99m Tc pertechnetate.(a)The Ad-CMV-GFP-infected tumor is not visible using99m Tc pertechnetate imaging.(b)The Ad-SUR-NIS-infected tumor is clearly visible,with an intensity comparable to that of the thyroid or the bladder.Targeted radioiodine therapy for tumorsR Huang et al 148Cancer Gene Therapyanimals that received 131I and Ad-CMV-GFP continued to grow until the end of the experiment.In contrast,tumors in animals that received injections of both Ad-SUR-NIS intratumorally and 131I intraperitoneally began to show a slight reduction in growth on day 10and day 15,and from day 20to day 30,tumor volumes in this group were statistically different from Ad-CMV-GFP-infected controls.The volume reduction rates at days 20,25and 30were 32.9±11.3%(P ¼0.029),46.2±4.4%(P ¼0.001)and 51.4±15.9%(P ¼0.012),respectively.These results suggest that expression of NIS in combina-tion with 131I treatment may prove to be a possible strategy for reduction of tumor growth in vivo .Membrane expression of NIS in tumors injected with Ad-SUR-NISFor in vivo experiments,expression of the NIS protein in the tumor was characterized by immunohistochemical staining.Tumors injected with Ad-SUR-NIS werespecifically labeled with anti-NIS beling was clearly confined to cell membranes,indicating that expression of NIS driven by the Ad-SUR-NIS virus leads to correct localization of the NIS protein in vivo .Representative tumor sections are shown in Figure 6.DiscussionConsistent with the role of NIS in the success of radioiodine therapy for thyroid cancer,targeted expres-sion of NIS provides the possibility of NIS-targeted radionuclide therapy for treatment of non-thyroidal tumors.27–29In contrast to other potential therapeutic genes,therapeutic use of NIS has several advantages.The potential function of NIS as a diagnostic gene allows non-invasive radioiodine imaging to confirm and localize functional NIS expression before proceeding to the application of a therapeutic 131I dose.NIS gene therapy may also be more effective due to a substantial bystander effect caused by treatment with the b -emitter 131I,which has a path length of up to 2–4mm,and can cause the death of NIS-expressing cells as well as neighboring non-transfected cells.30The ultimate goal of cancer therapy is to maximize cancer cell-specific cytotoxicity and minimize toxic side effects in non-malignant cells.Gene therapy using tissue-specific promoters provides a means of selectively targeting therapeutic genes to malignant cells.31Several cancer-specific targeting promoters are available for targeting of individual cancers,including the prostate specific antigen promoter (prostate cancer),the albumin enhancer and promoter (hepatocellular cancer),the CEA promoter (medullary thyroid cancer,colorectal cancer),the MUC-1promoter (breast cancer,pancreatic cancer)and the chromogranin A promoter (neuroendocrine tumors).These promoters are cancer-type specific,but are limited in scope.The expression of survivin is noted in common human cancers but not in normal adult tissues.As the increased survivin activity is controlled transcriptionally,it has been suggested that the survivin promoter might control the transgene expression in a cancer-specific manner.32,33Figure 5Therapeutic effects of Ad-SUR-NIS infection in combina-tion with 131I treatment in PC-3xenografts.Tumor size was assayed after 131I treatment in nude mice bearing tumors infected with either Ad-SUR-NIS or Ad-CMV-GFP.Growth of Ad-SUR-NIS-infected PC-3tumors was significantly retarded (*,**,***P o 0.05)at days 20,25and 30after injection of 111MBq 131I.Figure 6Representative immunostaining of NIS in tumors from nude mice.Note the distinct cell membrane staining of NIS in the PC-3tumor infected with Ad-SUR-NIS (a ).In contrast,the non-infected PC-3tumor is negative for NIS immunoreactivity (b ).Original magnification for each panel was Â400.Targeted radioiodine therapy for tumors R Huang et al149Cancer Gene Therapy。
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配套大肠 杆菌
Amersham 原核表达载体
Amersham 原核表达载体
Amersham 原核表达载体
Invitrogen 原核表达载体
Lucigen
原核表达 菌株
Stratagene 原核表达菌株
Qiagen
原核表达 载体
Qiagen
原核表达 载体
Invitrogen 基因载体
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BL21(DE3) BL21(DE3)plysS Rosetta(DE3) Rosetta(DE3)plysS Rosetta-gami(DE3) Rosetta-gami B(DE3)pLysS Orgami(DE3) OrgamiB(DE3) BLR(DE3) pTWIN1 pTYB11 pMXB10 pTXB1 ER2566 pGEX-6P-1 pGEX-4T-2 pGEX-4T-3 pRset-a C41(DE3) BL21 codonplus（DE3）RIPL TAGZyme pQE-2 pQE-Trisystem pOG44 pORF-lacZ pORF-HSV1tk pSP73 pUC18 pUC19 pBR322 pSP64 pBluescript II SK(+) SuperCos 1 pGAPZα A pPICZα A
荧光素酶实验具体步骤及说明
双荧光素酶实验具体步骤及说明设计理念在检测一个报告基因测量结果的变化时，实验的准确性常被一些客观实验因素影响，如：细胞数目不均一、细胞代次不统一、转染效率不一致等等。
引入一个内参报告基因，以此来标定检测用报告基因的测量结果，从而达到有效地减少实验误差的目的。
系统工作原理双荧光素酶报告系统包括两个报告基因，一个检测用报告基因A 与一个内参用报告基因B。
一般是将检测用报告基因A 序列与调控启动子序列偶连在一起，或将报告基因A 序列与待检序列串联，报告基因A 荧光强度的相对改变与偶联的调控启动子的活性或待检序列的改变相关。
内参用报告基因B 与一个组成型启动子偶联，这个启动子不受各种实验条件变化的干扰，所以报告基因B 的荧光为组成型表达，可以作为内参。
检测用报告基因A、内参用报告基因B 可以装在不同的载体上，共同转染细胞，通过这种方法，把可能削弱实验准确性的内在因素的变化降到最小。
有的载体上同时含有检测用报告基因A 和内参用报告基因B，这样使实验操作更加准确、简便、快捷。
常用载体（Promega ）实验用报告基因：pGL3 Luciferase Reporter Vectors （pGL3-Control Vector 、pGL3-Basic Vector 、pGL3-Promoter Vector、pGL3-Enhancer Vector）；psiCHECK TM Vector（psiCHECK TM -1 Vector、psiCHECK TM -2 Vector*）；pmirGLOVector*。
内参用报告基因（组成型表达海肾荧光素酶）：pRL-TK；pRL-SV40；pRL-CMV；pRL-Null。
注：psiCHECK TM -2 Vector*、pmirGLO Vector* 同时表达萤火虫荧光素酶（Firefly Lucifera se ）及海肾荧光素酶（Renilla Lucifera s e），故无需准备对照pRL 系列内参质粒，可直接进行下游双荧光素酶检测实验。
5、转录调控
分子机制研究套路（五）转录调控课题：转录因子A对B基因的转录调控1．概念介绍：转录水平的调控是真核生物基因表达调控中重要环节。
真核细胞RNA聚合酶自身对启动子并无特殊亲和力，单独不能进行转录，也就是说基因是无活性的。
因此，转录需要众多的转录因子和辅助转录因子形成复杂的转录装置。
在基因转录起始阶段，通用转录因子协助RNA聚合酶与启动子结合，但其作用很弱，不能高效率地启动转录。
只有在反式作用因子（基因特异性转录因子）的协助下,RNA聚合酶II和TFII才能有效地形成转录起始复合物。
反式作用因子（transactingfactor）在转录调节中具有特殊的重要性。
它是能直接或间接地识别或结合在顺式作用元件8~12bp核心序列上,参与调控靶基因转录效率的一组蛋白质。
这类DNA结合蛋白有多种，能特异性识别这类蛋白的序列也有多种，正是不同的DNA结合蛋白与不同的识别序列之间的空间结构上的相互作用，以及蛋白质与蛋白质之间的相互作用构成了复杂的基因转录调控机制的基础。
在真核生物中转录因子的调控是最重要，也是研究得最多的。
蛋白质相互作用在转录因子活性的调控方面具有重要的意义。
细胞内的反式作用因子都是处于有活性和无活性两种状态，这两种状态是可以转换的。
反式作用因子处于无活性状态时，与之相应的基因就不能表达；反式作用因子处于有活性状态、并与相应的顺式作用元件结合时，就可以促进RNA聚合酶和通用转录因子与相应的启动子结合，形成转录起始复合物。
所以，真核基因的表达调控主要是调节反式作用因子的活性，随后反式作用因子调控基因的转录起始。
转录因子被激活后，即可识别并结合上游启动子元件和增强子，对基因转录发挥调控作用。
大部分转录因子在激活以后与顺式作用元件结合，旦也可能有一些转录因子是先结合DNA,被激活后才发挥调节功能。
增强子和上游启动子元件可以结合一些相同的蛋白质，在不同的基因中，存在着同样的顺式作用元件，这表明一些数量有限的基因调控蛋白控制着真核细胞的基因表达。
常用载体通用引物
PBRforBam
PBRrevBam
pBR322(EcoRI/HindIII-sites)
pBRforEco
pBRrevHind
pBR325(BamHI-site)
PBRforBam
PBRrevBam
pBR329(BamHI-site)
PBRforBam
PBRrevBam
pDONR223
M13F
M13R
pDrive
T7/M13rev
SP6/M13for
pDsRED1-C1
(DsRed1-N-f)
DsRed1-C-r
pDsRed1-N1
CMV-Profor
RFP-Nrev,CMV-R?
pDsRed2-N1
(DsRed1-N-f)
DsRed1-C-r
pDsRed2-C1
BGH rev
pcDNA6/myc-His(A,B,C)
CMV-Profor,T7
BGH rev,c-mycrev
pcDNA6/V5-His(A,B,C)
CMV-Profor,T7
BGH rev,V5rev
pCEP4
pCEP-F/CMV-Profor
EBVrev
pCI
T7(17BASE)
EBVrev
T7,CMV-Profor
BGH rev,c-mycrev
pcDNA4/TO
TREfor/CMV-Profor
BGH rev
pcDNA4/V5-His(A,B,C)
CMV-Profor,T7
BGH rev,V5rev
pcDNA4-TET/0N/AMP+
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荧光素酶报告基因载体 —— pGL3
产品包装目录号价格
pGL3-Control Vector 20ug E1741
pGL3-Basic Vector 20ug E1751
pGL3-Promoter Vector 20ug E1761
pGL3-Enhancer Vector 20ug E1771
描述: 荧光素酶报告基因载体系列pGL3，为定量具有调节哺乳动物基因表达潜在能力的各种因子的活性提供一个分析基础。
这些可以是顺式或反式作用因子。
经重新设计，pGL2荧光素酶报告基因载体系列的骨架成为了pGL3系列载体的骨架，提高了表达量。
重新设计包括对萤火虫(Photinus pyralis)荧光素酶的编码区进行修饰，使其在被转染的真核细胞中能进行最优转录活性监控。
这个遗传报告基因的检测快速，灵敏，并且定量准确。
另外，荧光素酶报告基因载体系列具有各种不同特性，以帮助了解所研究的假设调节序列的结构特征。
特点：
●易于使用：位于luc+ 基因5'末端的Nco I 位点允许使用这个唯一的Nco I位点制造与报告基因融合的蛋白。
●灵活：可从位于多克隆位点区的Sma I 位点插入平端片段于克隆位点区，并被其它限制性内切酶从两边切下。
●多功能：在紧靠luc+ 基因下游创造的Xba I 位点可促进mRNA 3'不翻译区的插入或亚克隆荧光素酶基因。
操作手册：
技术手册： TM033
保存条件：-20℃，菌株保存在-70℃。
GenBank®/EMBL 提取号码：pGL3-Basic: U47295. pGL3-Control: U47296. pGL3-Enhancer: U47297. pGL3-Promoter: U47298.
注意：载体pGL3的特定转录特征随细胞类型的不同
而不同。
特别是对于含有SV40大T抗原的COS 细胞，更是如此。
SV40大T抗原促进从SV40复制起点的复制，这个复制起点位于pGL3-Promoter和pGL3-Control载体的启动子上。
大T 抗原与SV40复制起点的组合使得这些载体在COS 细胞中具有更高的拷贝数，反过来导致报告基因具有比其它细胞和载体组合更高的表达量。
荧光素酶报告基因载体系列pGL3提供SV40早期启动子和增强子序列的不同组合，以提供灵活的实验战略，方便使用者。
可通过巢式缺失获得遗传因子的功能图谱。
可为突变或测序制备单链DNA。
跨克隆位点两端测序和进行引物延伸分析所需的引物也可从普洛麦格公司得到。