Luciferase Reporters: Less is More

pGL4 Vectors: A New Generation of Luciferase Reporter Vectors

By Aileen Paguio, M.S., Brian Almond, Ph.D., Frank Fan, Ph.D., Pete Stecha, B.S., Denise Garvin, M.S., Monika Wood, M.S., and Keith Wood, Ph.D., Promega Corporation Abstract Table 1. pGL4 Vector Components. Reporter technologies are powerful tools by which to study Multiple and regulation (1,2). Among reporters used in Cloning Reporter Degradation Mammalian Vector Region Gene Sequence Promoter mammalian cells, the luciferases are preferred because they are pGL4.10[luc2]Yesluc2 No No highly sensitive, flexible, easy to quantify and detectable over a pGL4.11[luc2P] Yes “ hPEST No broad dynamic range (3). We have developed several new pGL4.12[luc2CP] Yes “ hCL1-hPEST No reporter designs for firefly and Renilla luciferases, which allow pGL4.13[luc2/SV40] No “ No SV40 a range of uses from measurement of transcriptional activity in pGL4.70[hRluc]YeshRluc No No cell biology applications to high-throughput screening in drug pGL4.71[hRlucP] Yes “ hPEST No discovery. The resulting vectors yield higher expression with pGL4.72[hRlucCP] Yes “ hCL1-hPEST No decreased signal-to-background ratios and respond more rapidly pGL4.73[hRluc/SV40] No “ No SV40 to transcriptional dynamics. The vectors are also designed to pGL4.74[hRluc/TK] No “ No HSV-TK reduce the risk of anomalous effects. pGL4.75[hRluc/CMV] No “ No CMV

Our goal was to create an ideal reporter that: these problems. We applied our “cleaning” strategy to i) expresses uniformly and optimally in host cells; the entire vector backbone, removing cryptic regulatory ii) minimizes off-target responses sequences where possible, while maintaining functionality. In addition to the redesigned vector (reduces anomalous expression); and backbone, the pGL4 Vectors incorporate a variety of iii) responds rapidly to transcriptional dynamics. features such as a choice of luciferases, including the Rapid Response™ Reporter versions, and promoterless (basic) and promoter-containing (control) vectors. Table 1 Introduction describes the components of 10 of the pGL4 Luciferase We have developed a new series of reporter vectors, the Reporter Vectors. A schematic diagram of a pGL4 Vector pGL4 Luciferase Reporter Vectors(a,b,c,d,e). In designing is shown in Figure 1. these vectors our goal was to create an ideal reporter that: i) expresses uniformly and optimally in host cells; Reduced Risk of Anomalous Expression ii) minimizes off-target responses (reduces anomalous The pGL3 Vector backbone was used as a template for expression); and iii) responds rapidly to transcriptional the pGL4 Vector backbone. Numerous consensus dynamics. To develop improved reporters from the binding sites are present in the native luciferases, we addressed these three criteria. pGL3 Vector backbone (Figure 2). To reduce the risk of anomalous expression and increase the reliability of First, we increased the expression levels of the luciferase reporter gene expression, we redesigned the backbone reporter in mammalian cells by redesigning the luciferase Synthetic poly(A) gene using codons preferred for mammalian sequence signal/transcriptional pause site expression (4,5). Second, we removed cryptic regulatory (for background r sequences, reducing the risk of anomalous expression Amp reduction) (4,6–8). Finally, we developed destabilized luciferase Bgl I/Sfi I 8 Acc65 I 14 (Rapid Response™ Reporters), with greatly improved Kpn I 18 EcoICR I 23 response rates compared to native luciferase (9). Sac I 25 ori pGL4.10[luc2] Nhe I 27 Vector Xho I 33 The vectors used to deliver reporter genes into host cells (4242bp) EcoR V 41 are also critical for the overall performance of the Bgl II 46 2025 Sal I Bgl I/Sfi I 59 reporter assay. Cryptic regulatory sequences such as 2019 BamH I Hind III 65

transcription factor binding sites and/or promoter SV40 late modules found on the vector backbone could lead to poly(A) signal Nco I 98 high backgrounds and anomalous responses (10,11). Hpa I 1917 luc2

Although this is a common issue for all mammalian Xba I 1757 4721MA reporter vectors, little has been done until now to rectify Figure 1. The pGL4.10[luc2] Vector.

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AD-b PXR/CAR/RXR Pbx-1 Nkx2.5 CCAAT Cut-like SRF Gfl-1B TALE STAT5 ELF-2 Cut-like RFX1 BRN-23 GBF3 CCAAT Prom L Gut OBF1 En-1 GBF3 VIS1 HEN1 SRF B-cell PAR Mamm.C GBF3 Elk-1 Sox-5 NMP4 NKX3.1 AREB6 Msx-1 Hox1.3 E2F C-Myb SRF Otx2 HNF 1 HLF NFAT Myo HNF1 AhR ATF4 LHX3 Pax2/5/8 AC-t STAT5 VIS1 VIS 1 ST/TA SF1 E2F CREB Lmo2 NFY MyT1 VIS 1 HMX3 NKX3.1 MIS HNF4 HNF1 SRF Lmo2 MEIS1 B-cell ST/TA MEF Mt TCF/LEF-1 HNF 1 MOK-2 CDE HNF3 GFI1 Pit1 OBF 1 E2F RP58 EBV GSh-1 Elk-1 FOXL1 KLF3 MIBP-1/RFX1 POZ CDF-1 FAST-1/S MAD Pbx1/Meis1 Neu1/3 CCAAT v-Myb COMP1 E2F MESI1 Pax-3 Tst-1/Oct-6 Lmo2 B-cell MEIS 1 E2F Ikaros 3 r CPBP WHP CBF 3 E4BP4 ST/TA GBF1 Pax1 CDE/CHR TCF/LEF-1 Arnt p Cone-rod MSX1/MSX-2 MyT1 m Pax-3 Ras C-Ets-1 A FLI PAR Pax1 Tax/CREB GBF3 AD-b CDP CCAAT C2HC1 Pbx1 HNF1 GABP E2 E4BP4 RFX1 Musc. Pax1 Oct1,2 NGN1/3 FAST-1/Smad Ikaros2 Mus v-Mar v-Myb Brn3 FOXF2 PAR B-cell NUDR ATF CCAAT CCAAT MIBP-1 MyT1NUDR EKLF Pbx1 TEF-1 OBF1 E4BP4 CDF-1 HOX C2HC SRE OBF1 Cut-like FOXF2 RFX1 OBF1 Gut NF-kB Elk-1 PAX9 AD-b CREB MEIS1 MEIS1 ST/TA C2H2 SOX-5 CCAAT GBF1 IS NRF2 Elf-2E2F v-Myb POZ GBF2 Pbx1 Brn-2 IRF-3 HIF-1 NF-kB luc+ MOK-2 Brn2 c-Ets-1 HLF BACH1 Neu-rGBF1 MMC CCAATHIF1 COMP1 VIS1 PBX/MEIS1 v-Myb Pit1 OBF1 Barb Ikaros3 HLF Pax1 ATF Tst-1/Oct-6 MEIS1 OBF1 STAT5 Neu-r ST/TA Brn23 E4BP4 COMP1 c-Myb MEF2 WTS MEIS1 SRF MOK-2 GATA RF-7 HoxC-Rel OBF1 FAST-1 CREB TATA Clox GBF1 POZ ST/TA POZ v-Myb c-Myb GLI-k HNF6 B-cell ZF GBF1MRE POZ My11 MEF TALE TCF/LEF-1 MMC RFX1 B-cell E2F v-Myb Albumin D-box HEN1 MIBP1 Pbx1 Cut-like Lmo2 Pro L S8 STs TTF1 Myo Smad4 C-r EG3 Myo E2F Tax FoxA2 PAX-6 RFX1 CDE/CHR ST/TA v-Myb HNF4 CREB GBF1 Bright c-Myb CHOP FLI ST/TA NKX3.1 WHP Cdf1 X-Box Pax-6 Tax/CREB Brn-3 Sox-5 c-Myb NF Y Ikaros2 FAST-1/SMAD NF-KB Se-CtRNA PXR/RXR/CAR Neu-r Gsh-1 E2F CCAAT NUDR NUDR PAR HLF MZF-1 Thing1 AP 2 HAND2 E2F Gfi-1B GBF1 MAZ TATA PAR E4BP4 NKX3.1 BPV CREB Neu1/3 HNF4 Lmo2 E2F c-Myb NMP4 Tal-1a TCF HNF1 AC-t CREB GLI-K PAX6 E4BP4 COMP1 C-r RFX1 IRF2 Cart-1 Pbx1/Meis1 PAR ori GBF1 Elk-1 NKX3.1 E2F PAX2/5/8 CCAAT Mamm.C P53FLI E2F TCF11 Nkx2.5 GBF3 CREB HAND2 NMP4 E4BP4 Pdx1 Cart-1 Avian C c-Myb -2 AB-D Tal-1a PAR MyT1 PAR MOK-2 NUR77/Nurr1/Nor-1 Brn-3 OBF-1 COMP1 RP58 TALE Pax-3 Cut-like Brn-3 Clox Pbx-1 Ikaros-3 ST/TA Cart-1 CDP Cut-like CREB FOXC1 AC-t Pbx-1 RAR E2FCREB Elk-1 Pbx1 Elk-1 E2F IF1 E2F C-Abl PAR GFI1 PR c-Myb CDP v-Myb CDF-1 ST/TA NUDR Pax1 SRF MyT1 Arnt CDP OBF1 NKX3.1 pGL3 Elk-1 AC-t Backbone

v-Myb NF-kB p53 Nco I

PL p53 Avian C PAX9 CDF-1 Pax TALE Hox-1.3 E4BP4 AD-b NKX3.1 VDR/RXR Dec1 HNF1 GBF1 Pax1 Bel-1 AD-b Avian C luc2 Baso r p E4BP4 NKX3.1 m FKHRL1 A Cart-1 PAX9 CDF1 LBP1c/LSF Mamm C c-Myb AP1 Cart-1 Runt-2 v-Myb CDF1 MIT/TFE NUDR TCF/LEF1 GBF1 Nkx2.5 Pdx-1 Baso Cdx-2 p53 Brn-3 Runt-2 FOXC1 Cart-1 ori OBF1 AD-b PAR 4760MA GBF3 RP58 Tal-1a COMP1 CDP Clox Figure 3. Comparison of consensus transcription factor binding sites in Avian C Pbx1 Pbx-1 luc+ and luc2 genes.

pGL4 IRF1 Backbone TEF-1 Improved Luciferase Expression

Nco I 4761MA The pGL4 Vectors also feature an improved firefly Figure 2. Comparison of consensus transcription factor binding sites in luciferase gene, luc2. Compared to its predecessor, luc+ pGL3 and pGL4 Vector backbones. in the pGL3 Vectors, luc2 is codon optimized and has fewer cryptic regulatory sequences (Figure 3). The gene of the pGL3 Vector region from the start of the reporter was synthetically redesigned by changing the codons to gene to the bacterial origin of replication sequence to those most frequently used in mammalian cells, while greatly reduce the number of consensus transcription simultaneously removing most of the consensus factor binding sites and thus the risk of anomalous sequences for transcription factor binding sites. expression (Figure 2). Other modifications included a Additionally, the number of predicted promoter modules redesign of the multiple cloning region, removal of the f1 have been reduced to a single module in the luc2 gene. origin of replication, deletion of an intronic sequence and In the transfection experiment shown in Figure 4, the reduction in the number of promoter modules. (Promoter synthetic firefly luciferase gene, luc2, displayed increased modules are regulatory elements consisting of two or expression compared to luc+. To ensure that the synthetic more transcription factor binding sites separated by a construction affected only expression, both the luc+ and spacer.) A synthetic poly(A) signal/transcriptional pause luc2 genes were cloned into the pGL3-Control Vector. The site was retained upstream of either the multiple cloning vectors were cotransfected with a control and, after 24 region (in promoterless vectors) or the mammalian hours, the relative light units measured and normalized promoter (in promoter-containing vectors). The bacterial to the control. When compared to luc+, the luc2 gene origin of replication and the SV40 late poly(A) signal demonstrated a 4.1- to 11.8-fold increase in expression downstream of the reporter gene were not modified. for the four mammalian cell lines tested. The newly designed pGL4 Vectors behave similarly in terms of replication, selection and maintenance in E. coli to their pGL3 counterparts, despite significant changes in the DNA sequences. 8 Table 2. Background Comparison Among the pGL4, pGL3 and phRL 10,000,000 Vectors. 4.9 s) Signal-to- Percent Increase t 1,000,000 1 luc+ Vector Background Ratio (pGL4 Vectors) 7.9 Uni pGL4.13[luc2/SV40] vs.pGL4.10[luc2] 3,162 ± 337 373 t luc2 pGL3-Control vs. pGL3-Basic 668 ± 57 100,000

ive Ligh 11.8 t

pGL4.73[hRluc/SV40] vs. pGL4.70[hRluc] 628 ± 78 3,205 Luminescence 4.1 10,000 phRL-SV40 vs. phRL-null 19 ± 1.7 (Rela

pGL4.74[hRluc/TK] vs. pGL4.70[hRluc] 79 ± 8 3,335 1,000 NIH/3T3 CHO HEK 293 HeLa phRL-TK vs. phRL-null 2.3 ± 0.9 4731MA Figure 4. The firefly luc2 gene displays higher expression than luc+. pGL4.75[hRluc/CMV] vs. pGL4.70[hRluc] 1,103 ± 115 6,388 The luc2 gene was cloned into the pGL3-Control Vector (Cat.# E1741), replacing the luc+ gene. Thus both firefly luciferase genes were in the same pGL3-Control Vector phRL-CMV vs. phRL-null 17 ± 1.5 backbone. The two vectors containing either of the firefly luciferase genes were 1To generate signal-to-background ratios the luciferase-containing vectors were transfected into NIH/3T3, CHO, HEK 293 and HeLa cells using the phRL-TK Vector transfected into CHO cells. At 24 hours post-transfection the cells were lysed, (Cat.# E6241) as a transfection control. Twenty-four hours post-transfection the cells luminescent signals were generated using the Dual-Luciferase® Assay System and were lysed with Passive Lysis Buffer (Cat.# E1941), and luminescence was measured the relative light units were corrected for transfection efficiency, yielding “normalized using the Dual-Luciferase® Assay System (Cat.# E1910). Relative light units were signals”. To calculate the signal-to-background ratio, the normalized signal from normalized to the Renilla luciferase expression from the phRL-TK Vector transfection transfection of the promoter-containing construct was divided by the normalized control. The fold increase in expression value is listed above each pair of bars. signal from the corresponding promoterless vector. The experiment was repeated in CHO, HeLa, NIH/3T3 and HEK 293 cells, generating similar results. Conclusion Improved Signal-to-Background Ratios The pGL4 Vector series represents a new generation of luciferase reporter vectors. There are a variety of gene Modifications to the pGL4 Vector backbone combined and promoter configurations to choose from. Compared with synthetic reporter genes have resulted in an to their predecessors, the pGL3 Vectors, the pGL4 Vectors improved signal-to-background ratio compared to their reduce the risk of anomalous expression, have better pGL3 and phRL Vector counterparts. In Table 2 these luciferase expression and improved signal-to- vectors are compared to their corresponding background ratios. These features plus the ease of promoterless controls and the percent increase in the transfer from one vector to another make the pGL4 signal-to-background ratios reported. At the low end, the Vectors the best choice in luciferase reporter vectors. pGL4.13[luc2/SV40] Vector displayed a 373% increase in signal-to-background ratio, while the greatest increase in Editor’s Note: Additional pGL4 Vectors are being signal-to-background ratio was demonstrated for the developed to assist with your reporter needs, including pGL4.75[hRluc/CMV] Vector at 6,388%. vectors containing selectable markers. Watch for the release of these and additional pGL4 Reporter Vectors Easy Transfer Between Vectors in 2005. Another feature of the pGL4 Vectors is the redesigned multiple cloning region for easy transfer of promoters References and other transcriptional elements from one vector to 1. Wood, K.V. (1995) Curr. Biol. 6, 50–8. another within the pGL4 Vector series or from pGL3 to 2. Naylor, L.H. (1999) Biochem. Pharmacol. 58, 749–57. pGL4 Vectors (Figure 5). The new multiple cloning 3. Wood, K.V. (2004) Progress in Biomedical Optics and Imaging, Nicolau, region retains most of the key restriction enzyme sites D.V. and Raghavachari, R., eds. SPIE–The International Society for from pGL3 Vectors, while introducing two Sfi I sites at Optical Engineering, Bellingham, 66–77. either end of the multiple cloning region (Sfi I recognizes 4. Zhuang, Y. et al. (2001) Promega Notes 79, 6–11. the DNA sequence GGCCNNNNNGGCC, where N is A, 5. Almond, B. et al. (2003) Promega Notes 85, 11–4. T, G or C). Each site generates unique cohesive ends. 6. Annicott, J.S. et al. (2001) BioTechniques 31, 993–4. This property allows directional transfer of elements 7. Ho, C.K.M. and Strauss, J.F. (2004) BMC Biotechnology 4, 10. (without an internal Sfi I site) from the multiple cloning 8. Grimm, S.L and Nordeen, S.K. (1999) BioTechniques 27, 220–2. region of one pGL4 Vector to another with a single Sfi I 9. Almond, B. et al. (2004) Promega Notes 87, 18–22. digestion. 10. Bert, A.G. et al. (2000) Plasmid 44, 173–82. 11. Thirunavukkarasu, K. et al. (2000) BioTechniques 28, 506–10.

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RVprimer3

5´...ACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCT

Synthetic poly(A) signal/transcriptional pause site

Sfi I Acc65 I EcolCR I Bgl I Kpn I Sac I Nhe I Xho I EcoR V Bgl II

GGCCTAACTGGCCGGTACCTGAGCTCGCTAGCCTCGAGGATATCAAGATCT

Sfi I Aileen Paguio, M.S. Brian Almond, Frank Fan, Ph.D. Bgl I Hind III Nco I Research Scientist Ph.D. Research Scientist Project Manager GGCCTCGGCGGCCAAGCTTGGCAATCCGGTACTGTTGGTAAAGCCACCATGG...3´

Luciferase start 4735MA Figure 5. The multiple cloning region of the pGL4 Vectors. The Bgl I and EcoR V sites in this figure are also present in the hRluc gene and should not be used for cloning into the hRluc- (Renilla-) based pGL4 Vectors.

Protocol ◆ pGL4 Luciferase Reporter Vectors Technical Manual #TM259, Promega Corporation. www.promega.com/tbs/tm259/tm259.html Pete Stecha, B.S. Denise Garvin, M.S. Monika Wood, M.S. Research Scientist Research Scientist Research Scientist Ordering Information Product Size Cat.# Price ($) pGL4.10[luc2] Vector 20µg E6651 288 pGL4.11[luc2P] Vector 20µg E6661 288 pGL4.12[luc2CP] Vector 20µg E6671 288 pGL4.13[luc2/SV40] Vector 20µg E6681 288 pGL4.70[hRluc] Vector 20µg E6881 288 pGL4.71[hRlucP] Vector 20µg E6891 288 pGL4.72[hRlucCP] Vector 20µg E6901 288 pGL4.73[hRluc/SV40] Vector 20µg E6911 288 Keith Wood, Ph.D. pGL4.74[hRluc/TK] Vector 20µg E6921 288 Research Fellow pGL4.75[hRluc/CMV] Vector 20µg E6931 288 (a) Patent Pending. (b) The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673. A license (from Promega for research reagent products and from The Regents of the University of California for all other fields) is needed for any commercial sale of nucleic acid contained within or derived from this product. (c) A limited license under Promega’s U.S. Pat. Appln. Ser. No. 09/645,706 and other pending patents is conveyed with the purchase of the pGL4 Luciferase Reporter Vectors. In addition, a limited license under Promega’s U.S. Pat. Appln. Ser. No. 10/664,341, PCT Pat. Appln. Ser. No. PCT/US03/28939 and various corresponding patent applications and issued patents is conveyed with the purchase of the following pGL4 Vectors: Cat.# E6661, E6671, E6891 and E6901. For Nonprofit Organization research use only. With respect to research use by for-profit organizations, or any diagnostic or therapeutic uses, please contact Promega Corporation for supply and licensing information. (d) Licensed from University of Georgia Research Foundation, Inc., under U.S. Pat. Nos. 5,292,658, 5,418,155, Canadian Pat. No. 2,105,984 and related patents. (e) The CMV promoter and its use are covered under U.S. Pat. Nos. 5,168,062 and 5,385,839 owned by the University of Iowa Research Foundation, Iowa City, Iowa, and licensed FOR RESEARCH USE ONLY. Commercial users must obtain a license to these patents directly from the University of Iowa Research Foundation. Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information. Dual-Luciferase is a registered trademark of Promega Corporation. Rapid Response is a trademark of Promega Corporation.

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