一、Pathway reporter简介:细胞受到刺激时会做出一系列的反应,包括细胞增殖、分化、凋亡和炎症反应等,转录因子(Transcription factor, TF)在这些过程中发挥核心作用, 检测其活性水平的改变将直接联系到胞内信号通路的状况,有助于揭示细胞反应的分子机制。Geneup公司设计的信号通路报告载体,可以快速灵敏地检测转录因子的活性,对信号通路进行高通量筛选,也可以对特定信号通路进行实时监测。
1.普通质粒表达载体:pTRE-F.Luc载体特征:
- 在转录调控因子结合位点(TRE)上游插入了人造poly(A)终止信号,可以有效的降低背景;
- 利用萤火虫荧光素酶基因(Fluc)可对信号活性做出准确的定量分析;
- 利用Hygromycin抗性基因筛选稳定表达的细胞系。
2. 慢病毒表达载体:pL-TRE-F.Luc 载体特征: - 反向克隆TRE-pA-Fluc.copGFP表达框,可有效减少因LTR启动子造成的背景;
- 利用萤火虫荧光素酶基因(Fluc)可对信号活性做出准确的定量分析;
- 通过copGFP绿色荧光标记蛋白可实现对信号通路活性的快速观测;
- 可以直接转染,也可包装成病毒后感染;
- 利用Puromycin抗性基因筛选稳定表达的细胞系。
Pathway reporter实例
1. NFAT-FLuc
转染后24h用10ng/mL PMA和1µM ionomycin处理细胞,用Luciferase Assay进行检测。
2. NFkB-Fluc2
转染后24h用50ng/mL TNF-α处理细胞,用Luciferase Assay进行检测。
3. pTGFβ-Fluc2
转染后24h用25ng/µL TGFβ处理细胞,用Luciferase Assay进行检测。
4. pAP-1-Fluc2
转染后24h用10ng/mL PMA处理细胞,用Luciferase Assay进行检测。
5. HIF1a-Fluc2
转染后24h用250µM CoCl
2处理细胞8h,用Luciferase Assay进行检测。
Pathway reporter载体 1. pTRE-Fluc2系列:
已完成45个pathway reporter,详情见下表:
Vector Name | Pathway | Transcription Factor(TF) |
pAhR-Fluc2 | Xenobiotic | Xenobiotic Response Element |
pAR-Fluc2 | Androgen | Androgen Receptor |
pAP-1-Fluc2 | MAPK/JNK | AP-1 |
pATF4/3/2-Fluc2 | Amino Acid Deprivation | ATF4/3/2 |
pATF6-Fluc2 | ATF6 | ATF6 |
pC/EBP-Fluc2 | C/EBP | C/EBP |
pCBF/NF-Y/YY1-Fluc2 | ER Stress | CBF/NF-Y/YY1 |
pCREB-Fluc2 | cAMP/PKA | CREB |
pE2F/DP1-Fluc2 | Cell Cycle | E2F/DP1 |
pEGR1-Fluc2 | EGR1 | EGR1 |
pElk-1/SRF-Fluc2 | MAPK/ERK | Elk-1/SRF |
pER-Fluc2 | Estrogen Receptor | Estrogen Receptor (ER) |
pFOXO-Fluc2 | PI3K/AKT | FOXO |
pGATA-Fluc2 | GATA | GATA |
pGLI-Fluc2 | Hedgehog | GLI |
pGR-Fluc2 | Glucocorticoid Receptor | Glucocorticoid Receptor (GR) |
pHNF4-Fluc2 | Hepatocyte Nuclear Factor 4 | HNF4 |
pHSF-Fluc2 | Heat Shock Response | HSF |
pHIF-1-Fluc2 | Hypoxia | Hypoxia-inducible factor-1 (HIF-1) |
pIRF1-Fluc2 | Interferon Regulation | IRF1 |
pKLF4-Fluc2 | KLF4 | KLF4 |
pLXRa-Fluc2 | Liver X Receptor | LXRa |
pMEF2-Fluc2 | MEF2 | MEF2 |
pMTF1-Fluc2 | Heavy Metal Stress | MTF1 |
pMyc/Max-Fluc2 | c-myc | Myc/Max |
pNanog-Fluc2 | Nanog | Nanog |
pNFAT-Fluc2 | PKC/Ca++ | NFAT |
pNFκB-Fluc2 | NFκB | NFκB |
pNrf2 & Nrf1-Fluc2 | Antioxidant Response | Nrf2 & Nrf1 |
pOct4-Fluc2 | Oct4 | Oct4 |
pp53-Fluc2 | p53/DNA Damage | p53 |
pPax6-Fluc2 | Pax6 | Pax6 |
pPPAR-Fluc2 | PPAR | PPAR |
pProgesterone Receptor-Fluc2 | Progesterone | Progesterone Receptor |
pRBP-Jk-Fluc2 | Notch | RBP-Jk |
pRAR-Fluc2 | Retinoic Acid Receptor | Retinoic Acid Receptor (RAR) |
pRetinoid X Receptor-Fluc2 | Retinoid X Receptor | Retinoid X Receptor |
pSMAD2/SMAD3/SMAD4-Fluc2 | TGFβ | SMAD2/SMAD3/SMAD4 |
pSox2-Fluc2 | Sox2 | Sox2 |
pSP1-Fluc2 | SP1 | SP1 |
pSTAT1/STAT1-Fluc2 | Interferon Gamma | STAT1/STAT1 |
pSTAT1/STAT2-Fluc2 | Type I Interferon | STAT1/STAT2 |
pSTAT3-Fluc2 | STAT3 | STAT3 |
pTCF/LEF-Fluc2 | Wnt | TCF/LEF |
pVitamin D Receptor-Fluc2 | VDRE | Vitamin D Receptor |
pCMV-Fluc2-Negative Control | | |
pFluc2-Positive Control | | |
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注明:如需包装慢病毒报告载体,请联系本公司销售部。
2. pL-TRE-Luc.copGFP系列:
已完成的pathway reporter见下表:
Vector Name | Pathway | Transcription Factor(TF) |
pL-AhR-Fluc2 | Xenobiotic | Xenobiotic Response Element |
pL-AR-Fluc2 | Androgen | Androgen Receptor |
pL-AP-1-Fluc2 | MAPK/JNK | AP-1 |
pL-ATF4/3/2-Fluc2 | Amino Acid Deprivation | ATF4/3/2 |
pL-ATF6-Fluc2 | ATF6 | ATF6 |
pL-C/EBP-Fluc2 | C/EBP | C/EBP |
pL-CBF/NF-Y/YY1-Fluc2 | ER Stress | CBF/NF-Y/YY1 |
pL-CREB-Fluc2 | cAMP/PKA | CREB |
pL-E2F/DP1-Fluc2 | Cell Cycle | E2F/DP1 |
pL-EGR1-Fluc2 | EGR1 | EGR1 |
pL-Elk-1/SRF-Fluc2 | MAPK/ERK | Elk-1/SRF |
pL-ER-Fluc2 | Estrogen Receptor | Estrogen Receptor (ER) |
pL-FOXO-Fluc2 | PI3K/AKT | FOXO |
pL-GATA-Fluc2 | GATA | GATA |
pL-GLI-Fluc2 | Hedgehog | GLI |
pL-GR-Fluc2 | Glucocorticoid Receptor | Glucocorticoid Receptor (GR) |
pL-HNF4-Fluc2 | Hepatocyte Nuclear Factor 4 | HNF4 |
pL-HSF-Fluc2 | Heat Shock Response | HSF |
pL-HIF-1-Fluc2 | Hypoxia | Hypoxia-inducible factor-1 (HIF-1) |
pL-IRF1-Fluc2 | Interferon Regulation | IRF1 |
pL-KLF4-Fluc2 | KLF4 | KLF4 |
pL-LXRa-Fluc2 | Liver X Receptor | LXRa |
pL-MEF2-Fluc2 | MEF2 | MEF2 |
pL-MTF1-Fluc2 | Heavy Metal Stress | MTF1 |
pL-Myc/Max-Fluc2 | c-myc | Myc/Max |
pL-Nanog-Fluc2 | Nanog | Nanog |
pL-NFAT-Fluc2 | PKC/Ca++ | NFAT |
pL-NFκB-Fluc2 | NFκB | NFκB |
pL-Nrf2 & Nrf1-Fluc2 | Antioxidant Response | Nrf2 & Nrf1 |
pL-Oct4-Fluc2 | Oct4 | Oct4 |
pL-p53-Fluc2 | p53/DNA Damage | p53 |
pL-Pax6-Fluc2 | Pax6 | Pax6 |
pPPAR-Fluc2 | PPAR | PPAR |
pL-Progesterone Receptor-Fluc2 | Progesterone | Progesterone Receptor |
pL-RBP-Jk-Fluc2 | Notch | RBP-Jk |
pL-RAR-Fluc2 | Retinoic Acid Receptor | Retinoic Acid Receptor (RAR) |
pL-Retinoid X Receptor-Fluc2 | Retinoid X Receptor | Retinoid X Receptor |
pL-SMAD2/SMAD3/SMAD4-Fluc2 | TGFβ | SMAD2/SMAD3/SMAD4 |
pL-Sox2-Fluc2 | Sox2 | Sox2 |
pL-SP1-Fluc2 | SP1 | SP1 |
pL-STAT1/STAT1-Fluc2 | Interferon Gamma | STAT1/STAT1 |
pL-STAT1/STAT2-Fluc2 | Type I Interferon | STAT1/STAT2 |
pL-STAT3-Fluc2 | STAT3 | STAT3 |
pL-TCF/LEF-Fluc2 | Wnt | TCF/LEF |
pL-Vitamin D Receptor-Fluc2 | VDRE | Vitamin D Receptor |
pL-Fluc2-Negative Control | | |
pL-Fluc2-Positive Control | | |
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温馨提示:不可用于临床ZL。