- Email: [email protected]
A new luciferase promoter insertion vector for the analysis of weak transcriptional activities
Gene, 124 (1993) 137-138 0 1993 Elsevier Science Publishers
B.V. All rights reserved.
137
0378-l 119/93/$06.00
GENE06900
A new luciferase promoter insertion vector for the analysis of weak transcriptional activities* (Recombinant DNA; neomycin resistance; transcription c-mos oncogene)
Rainer de Martin, John Strasswimmer
termination; vector construction; gene regulation; murine gas-l;
and Lennart
Philipson
European Molecular Biology Laboratory, D-6900 Heidelberg, Germany. Tel. 6221-387-200 Received by H. van Ormondt:
10 July 1992; Revised/Accepted:
22 September/24
September
1992; Received at publishers:
26 October
1992
SUMMARY
A new luciferase-encoding expression vector was generated by inserting the strong transcription termination signal from the mouse c-mos oncogene upstream from a multiple cloning site. This construct significantly reduced background transcription in NIH3T3 cells and has proven useful in the study of a weak promoter from the murine growth-arrestspecific gene gas-l.
The coding region of the firefly luc has been widely used as a reporter gene in analysing the regulation of transcription in eukaryotic genes. An easy-to-use highsensitivity enzymatic assay together with the absence of endogenous Luc activity in eukaryotes makes it a favorable system to investigate the transcriptional activity of promoter fragments in both transient and stable transfection experiments (De Wet et al., 1987). However, in some cell lines high background levels of Luc from the promoterless vector can be observed, apparently due to spurious transcription originating at cryptic promoter sites located in the upstream vector sequence. This background transcription may limit the analysis of weak promoters in certain cell types. Correspondence to: Dr. R. de Martin at his present address: Vienna International Research Cooperation Center (VIRCC), Brunnerstr. 59, A-1235 Wien, Austria. Tel. (43-l) 86634-620; Fax (43-l) 86634-623. * On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note. Abbreviations: bp, base pair(s); gas, growth-arrest-specific; kb, kilobase(s) or 1000 bp; Luc, luciferase; luc, Luc-encoding gene; MCS, multiple cloning site; neo, gene coding for neomycin resistance; nt, nucleotide(s); PCR, polymerase chain reaction; TK, gene encoding thymidine kinase; UMS, upstream mouse sequence.
We have solved this problem by constructing a new luc promoter insertion vector, pUBT-luc. It carries a strong transcriptional terminator, the upstream mouse sequence (UMS) from the murine c-mos gene (McGeady et al., 1986) before the MCS (Fig.1). The l.l-kb UMS fragment was generated by PCR from the plasmid pEU-cat (Piaggio and De Simone, 1990) with Sac1 and Not1 sites at its 5’ and 3’ ends, respectively, and cloned into PBS-luc (Francis-Lang et al., 1992). In addition, we have inserted the neo gene under the control of the herpes simplex 7X promoter from plasmid pMCl-neo (Thomas and Capecchi, 1987) to serve both as a selectable marker in stable transformants and as an internal control in RNase protection experiments. Plasmid pUBT-luc has enabled us to detect the very weak basal transcription from a 0.3-kb fragment of the gas-l promoter (Schneider et al., 1988) against a low background of luc expression in growing NIH3T3 fibroblasts. The background level is approx. threefold higher in the parental plasmid PBS-luc (Fig.2) and therefore masks the expression from the 0.3-kb gas-l fragment. The overall expression in the new plasmid carrying the gas-l promoter is slightly higher than that of the parental plasmid with gas-l. This small increase may be due to a lack
138 Sad
\ ,Sall 10000
s : ‘c ; 4
so00
woo 4000
2000
0
1
mHl,fill-in/Hincll) \Xhol Fig. I. Structure of the pUBT plasmid vector. Boxes represent the transcriptional terminator, UMS. the luc gene, the SV40 polyadenylation signal (po[yA) and the bacterial neo gene under the control of the herpes simplex TK promoter (7X-neo). Standard techniques were employed for construction. Promoter fragments can be inserted into a NotI-, SpeI-. Smal-, or HindIII-linearized or double-digested vector. For transfection, the construct can be linearized with SacI, Sal1 or KpnI (the transcriptional terminator is downstream from the KpnI site of the UMS sequence).
of interference of transcripts originating in the vector sequence of the unmodified plasmid with factors binding to the promoter region. We believe that this new vector will help in analysing the transcriptional activity of other weak promoters.
2
3
4
Fig. 2. Levels of Luc in growing NIH3T3 cells after transfection with different luc expression vectors. Twenty pg of luc construct (and 5 kg of a IacZ expression vector as internal control) were transfected using the BES method (Chen and Okayama. 1987) and Luc and p-galactosidase activities determined three days tal PBS-luc with a 0.3-kb NotI-SmaI 3, pUBT-luc; 4, pUBT-luc with the ment inserted. Values are expressed ized for P-galactosidase.
later. 1, parental PBS-luc; 2, parengas-l promoter fragment inserted; same 0.3-kb gas-l promoter fragas Luc relative light units normal-
REFERENCES Chen, C. and Okayama, H.: High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7 (1987) 2745-2752. De Wet, J.R., Wood, K.V., DeLuca, M., Helinski, D.R. and Subramani, S.: Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7 (1987) 725-737. Francis-Lang, H.. Price, M., Polycarpou-Schwarz, M. and DiLauro, R.: Cell-type-specific expression of the rat thyroperoxidase promoter indicates common mechanisms for thyroid-specific gene expesssion. Mol. Cell. Biol. I2 (1992) 576-588. McGeady, M.L., Wood, T.G., Maizel, J.V. and Van de Woude, G.F.: Sequences upstream from the mouse c+nos oncogene may function as a transcription termination signal. DNA 5 (1986) 289-298. Piaggio, G. and De Simone, V.: A new expression vector to study weak
ACKNOWLEDGEMENTS
promoters. Focus 12 (1990) 83-84. Schneider, C., King, R.M. and Philipson, L.: Genes specifically expressed at growth arrest of mammalian cells. Cell 54 (1988) 787-793.
We thank V. DeSimone for pEU-cat and H. FrancisLang for PBS-luc.
Thomas, K.R. and Capecchi, M.R.: Site-directed targeting in mouse embryo-derived stem 503-512.
mutagenesis by gene cells. Cell 51 (1987)
B.V. All rights reserved.
137
0378-l 119/93/$06.00
GENE06900
A new luciferase promoter insertion vector for the analysis of weak transcriptional activities* (Recombinant DNA; neomycin resistance; transcription c-mos oncogene)
Rainer de Martin, John Strasswimmer
termination; vector construction; gene regulation; murine gas-l;
and Lennart
Philipson
European Molecular Biology Laboratory, D-6900 Heidelberg, Germany. Tel. 6221-387-200 Received by H. van Ormondt:
10 July 1992; Revised/Accepted:
22 September/24
September
1992; Received at publishers:
26 October
1992
SUMMARY
A new luciferase-encoding expression vector was generated by inserting the strong transcription termination signal from the mouse c-mos oncogene upstream from a multiple cloning site. This construct significantly reduced background transcription in NIH3T3 cells and has proven useful in the study of a weak promoter from the murine growth-arrestspecific gene gas-l.
The coding region of the firefly luc has been widely used as a reporter gene in analysing the regulation of transcription in eukaryotic genes. An easy-to-use highsensitivity enzymatic assay together with the absence of endogenous Luc activity in eukaryotes makes it a favorable system to investigate the transcriptional activity of promoter fragments in both transient and stable transfection experiments (De Wet et al., 1987). However, in some cell lines high background levels of Luc from the promoterless vector can be observed, apparently due to spurious transcription originating at cryptic promoter sites located in the upstream vector sequence. This background transcription may limit the analysis of weak promoters in certain cell types. Correspondence to: Dr. R. de Martin at his present address: Vienna International Research Cooperation Center (VIRCC), Brunnerstr. 59, A-1235 Wien, Austria. Tel. (43-l) 86634-620; Fax (43-l) 86634-623. * On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note. Abbreviations: bp, base pair(s); gas, growth-arrest-specific; kb, kilobase(s) or 1000 bp; Luc, luciferase; luc, Luc-encoding gene; MCS, multiple cloning site; neo, gene coding for neomycin resistance; nt, nucleotide(s); PCR, polymerase chain reaction; TK, gene encoding thymidine kinase; UMS, upstream mouse sequence.
We have solved this problem by constructing a new luc promoter insertion vector, pUBT-luc. It carries a strong transcriptional terminator, the upstream mouse sequence (UMS) from the murine c-mos gene (McGeady et al., 1986) before the MCS (Fig.1). The l.l-kb UMS fragment was generated by PCR from the plasmid pEU-cat (Piaggio and De Simone, 1990) with Sac1 and Not1 sites at its 5’ and 3’ ends, respectively, and cloned into PBS-luc (Francis-Lang et al., 1992). In addition, we have inserted the neo gene under the control of the herpes simplex 7X promoter from plasmid pMCl-neo (Thomas and Capecchi, 1987) to serve both as a selectable marker in stable transformants and as an internal control in RNase protection experiments. Plasmid pUBT-luc has enabled us to detect the very weak basal transcription from a 0.3-kb fragment of the gas-l promoter (Schneider et al., 1988) against a low background of luc expression in growing NIH3T3 fibroblasts. The background level is approx. threefold higher in the parental plasmid PBS-luc (Fig.2) and therefore masks the expression from the 0.3-kb gas-l fragment. The overall expression in the new plasmid carrying the gas-l promoter is slightly higher than that of the parental plasmid with gas-l. This small increase may be due to a lack
138 Sad
\ ,Sall 10000
s : ‘c ; 4
so00
woo 4000
2000
0
1
mHl,fill-in/Hincll) \Xhol Fig. I. Structure of the pUBT plasmid vector. Boxes represent the transcriptional terminator, UMS. the luc gene, the SV40 polyadenylation signal (po[yA) and the bacterial neo gene under the control of the herpes simplex TK promoter (7X-neo). Standard techniques were employed for construction. Promoter fragments can be inserted into a NotI-, SpeI-. Smal-, or HindIII-linearized or double-digested vector. For transfection, the construct can be linearized with SacI, Sal1 or KpnI (the transcriptional terminator is downstream from the KpnI site of the UMS sequence).
of interference of transcripts originating in the vector sequence of the unmodified plasmid with factors binding to the promoter region. We believe that this new vector will help in analysing the transcriptional activity of other weak promoters.
2
3
4
Fig. 2. Levels of Luc in growing NIH3T3 cells after transfection with different luc expression vectors. Twenty pg of luc construct (and 5 kg of a IacZ expression vector as internal control) were transfected using the BES method (Chen and Okayama. 1987) and Luc and p-galactosidase activities determined three days tal PBS-luc with a 0.3-kb NotI-SmaI 3, pUBT-luc; 4, pUBT-luc with the ment inserted. Values are expressed ized for P-galactosidase.
later. 1, parental PBS-luc; 2, parengas-l promoter fragment inserted; same 0.3-kb gas-l promoter fragas Luc relative light units normal-
REFERENCES Chen, C. and Okayama, H.: High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7 (1987) 2745-2752. De Wet, J.R., Wood, K.V., DeLuca, M., Helinski, D.R. and Subramani, S.: Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7 (1987) 725-737. Francis-Lang, H.. Price, M., Polycarpou-Schwarz, M. and DiLauro, R.: Cell-type-specific expression of the rat thyroperoxidase promoter indicates common mechanisms for thyroid-specific gene expesssion. Mol. Cell. Biol. I2 (1992) 576-588. McGeady, M.L., Wood, T.G., Maizel, J.V. and Van de Woude, G.F.: Sequences upstream from the mouse c+nos oncogene may function as a transcription termination signal. DNA 5 (1986) 289-298. Piaggio, G. and De Simone, V.: A new expression vector to study weak
ACKNOWLEDGEMENTS
promoters. Focus 12 (1990) 83-84. Schneider, C., King, R.M. and Philipson, L.: Genes specifically expressed at growth arrest of mammalian cells. Cell 54 (1988) 787-793.
We thank V. DeSimone for pEU-cat and H. FrancisLang for PBS-luc.
Thomas, K.R. and Capecchi, M.R.: Site-directed targeting in mouse embryo-derived stem 503-512.
mutagenesis by gene cells. Cell 51 (1987)