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A series of mammalian expression vectors and characterisation of their expression of a reporter gene in stably and transiently transfected cells

A series of mammalian expression vectors and characterisation of their expression of a reporter... Downloaded from https://academic.oup.com/nar/article/18/4/1068/1112505 by DeepDyve user on 14 August 2020 © Oxford University Press 1990 1068 Nucleic Acids Research, Vol 18, No. 4 A series of mammalian expression vectors and characterisation of their expression of a reporter gene in stably and transiently transfected cells Jay P.Morgenstem* and Hartmut Land Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, UK Submitted January 24, 1990 The pJfi vector series has been designed to allow easy insertion pJQ Vectors and subsequent expression of exogenous genes in a wide variety of mammalian cells. The vectors share a common structure of a mammalian transcription unit composed of a promoter flanked 3 ' by a polyhnker, an intron, and a transcriptional termination signal which is linked to a pBR 322 derived backbone as shown. Each of the six vectors possesses a different promoter: pJ30, the SV40 early promoter (1); pJ4fi: the Mo MuLV LTR (2); pJ5O: the MMTV LTR (3); pJ5Efl: an MSV enhancer linked NIH 3T3 HeLa -1 5 ' to the MMTV LTR (4); pJ6fl: the rat /3 actin promoter (5); and pJ70- the SCMV IE94 promoter (6). To characterise the expression capabilities of these vectors in mammalian cells, the cat gene was inserted into each of the D •>•»• vectors and the resultant constructs were introduced transiently 10 1 and stably (with pSV2 neo as a selection marker) into NIH 3T3 and HeLa cells by calcium phosphate transfection. 1 T, 1 1 . AW All vectors led to high levels of stable and transient cat activity as well as dexamethasone inducibility in the cases of pJ5fi and s * s pJ5OE. Moreover, a striking difference in transient versus stable * 4 expression by pJ3flc<z/ and pJlQcat was noted in NIH 3T3 but not in HeLa cells. The actin promoter in pJ6fl has also been shown to be expressed efficiently in undifferentiated teratocarcinoma cells in which transcription from all other promoters mentioned here is severly repressed. For example, in contrast to drug resistance markers under control of viral promoters pJ6ilhygro can confer hygromycin resistance to these cells. REFERENCES 1 Howani.B H. (1983) Trends in Biochem Sa 8, 209-212 2 Shirauck.T M , Lemcr.R A. and SutdiffeJ G (1981) Nature 293, 543-548 3 Lee.F , Mulligan,R C , Berg,P and Ringold.G. (1981) Nature 294, 228-232 . 4 Uununs.L A , Gruss.P , PozzatU.R. and Khoury.G (1984) J Virol 49, 183-189 5 Nudel.U., Zakut.R , Shani.M., Neuman,S., Levy.Z and Yaffe,D (1983) Nud. Acids Res. 11, 1759-1771 6. Jeang.K -T , Rawlins.D R , Rosenfeld,P J , SheroJ H , Kelly, T J. and Hayward,G S (1987)7 Virol 61, 1559-1570 •Present address- Immunologic Pharmaceutical, 1 Kendall Square, Cambridge, MA 02139, USA http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

A series of mammalian expression vectors and characterisation of their expression of a reporter gene in stably and transiently transfected cells

Morgenstern, Jay, P.; Land,, Hartmut
Nucleic Acids Research , Volume 18 (4) – Feb 25, 1990
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Publisher
Oxford University Press
Copyright
© 1990 Oxford University Press
ISSN
0305-1048
eISSN
1362-4962
DOI
10.1093/nar/18.4.1068
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Abstract

Downloaded from https://academic.oup.com/nar/article/18/4/1068/1112505 by DeepDyve user on 14 August 2020 © Oxford University Press 1990 1068 Nucleic Acids Research, Vol 18, No. 4 A series of mammalian expression vectors and characterisation of their expression of a reporter gene in stably and transiently transfected cells Jay P.Morgenstem* and Hartmut Land Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, UK Submitted January 24, 1990 The pJfi vector series has been designed to allow easy insertion pJQ Vectors and subsequent expression of exogenous genes in a wide variety of mammalian cells. The vectors share a common structure of a mammalian transcription unit composed of a promoter flanked 3 ' by a polyhnker, an intron, and a transcriptional termination signal which is linked to a pBR 322 derived backbone as shown. Each of the six vectors possesses a different promoter: pJ30, the SV40 early promoter (1); pJ4fi: the Mo MuLV LTR (2); pJ5O: the MMTV LTR (3); pJ5Efl: an MSV enhancer linked NIH 3T3 HeLa -1 5 ' to the MMTV LTR (4); pJ6fl: the rat /3 actin promoter (5); and pJ70- the SCMV IE94 promoter (6). To characterise the expression capabilities of these vectors in mammalian cells, the cat gene was inserted into each of the D •>•»• vectors and the resultant constructs were introduced transiently 10 1 and stably (with pSV2 neo as a selection marker) into NIH 3T3 and HeLa cells by calcium phosphate transfection. 1 T, 1 1 . AW All vectors led to high levels of stable and transient cat activity as well as dexamethasone inducibility in the cases of pJ5fi and s * s pJ5OE. Moreover, a striking difference in transient versus stable * 4 expression by pJ3flc<z/ and pJlQcat was noted in NIH 3T3 but not in HeLa cells. The actin promoter in pJ6fl has also been shown to be expressed efficiently in undifferentiated teratocarcinoma cells in which transcription from all other promoters mentioned here is severly repressed. For example, in contrast to drug resistance markers under control of viral promoters pJ6ilhygro can confer hygromycin resistance to these cells. REFERENCES 1 Howani.B H. (1983) Trends in Biochem Sa 8, 209-212 2 Shirauck.T M , Lemcr.R A. and SutdiffeJ G (1981) Nature 293, 543-548 3 Lee.F , Mulligan,R C , Berg,P and Ringold.G. (1981) Nature 294, 228-232 . 4 Uununs.L A , Gruss.P , PozzatU.R. and Khoury.G (1984) J Virol 49, 183-189 5 Nudel.U., Zakut.R , Shani.M., Neuman,S., Levy.Z and Yaffe,D (1983) Nud. Acids Res. 11, 1759-1771 6. Jeang.K -T , Rawlins.D R , Rosenfeld,P J , SheroJ H , Kelly, T J. and Hayward,G S (1987)7 Virol 61, 1559-1570 •Present address- Immunologic Pharmaceutical, 1 Kendall Square, Cambridge, MA 02139, USA

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Nucleic Acids Research – Oxford University Press

Published: Feb 25, 1990

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