Versatile vectors for pulsed expression in eukaryotic cells

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Versatile vectors for pulsed expression in eukaryotic cells

Jocelyne Rech, Philippe Fort

To cite this version:

Jocelyne Rech, Philippe Fort. Versatile vectors for pulsed expression in eukaryotic cells. Nucleic

Acids Research, Oxford University Press, 1989, 17 (7), pp.2874-2874. �10.1093/nar/17.7.2874�. �hal-

02267534�

Volume 17 Number 6 1989 Nucleic Acids Research

Versatile vectors for pulsed expression in eukaryotic cells Jocelyne Rech and Philippe Fort*

UA CNRS 1191. Laboratoire de Biologie Moleculaire. Universit6 des Sciences et Techniques du Languedoc, 34060 Montpellier Cedex and Laboratoire de Biochimie. Centre Paul Lamarque. 34094 Montpellier Cedex.

France

Submitted January 14, 1989

We report here the construction of two vectors for transient expression of foreign genes in eukaryotic cells. Transiency of expression relies on the

of

c-fos SRE-promoter region (1) which can be transiently and efficiently induced by

wide set of stimuli (2), whereas its basal transcription is undetectable. A polylinker containing multiple restriction sites allows rapid and directed gene cloning at position -10 with respect to the cap site. Different 3' regions

are inserted downstream the polylinker: pFL3G contains a 460 bp BglII-PvuIl fragment from the rabbit 13-globin gene (3), and pFL3F contains a I kb SalI-BamHI fragment from the mouse

fos gene (1). This latter region confers a rapid turnover to a foreign RNA when appended 3' to it (Bonnieu et al, submitted). Both 3' regions contain signals for termination of transcription and polyadenylation. A schematic representation of pFL3G and pFL3F is shown below. These vectors

tested for 13-globin gene expression in mouse Ltk- cells: The coding region of the rabbit B3-globin gene was inserted in both vectors, leading to pFglo3G and pFglo3F. Stably transfected cells were serum deprived for 24 hours, then stimulated for growth upon 10% fetal calf serum addition as described (4). The time course of globin RNA accumulation arising from the transfected constructs is shown below.

3-Fos

3'GlobinHindlil PLS2

PL5_PLS

1~~~~~~~~PL

G~~~~~~~~~~'lobin

SRE/P1o5t SRE/PfOS

Hindlll

Smal pFL3G)) Smal pFL3F

\

lJC19 \4 < pUCI9

PLS1:BanrilI.XbaI.Sall.EcoRI.Stul.Xhol.Sstl.Nsil.BglIt PLS2:BamHI.XbaI.Sall.EcoRI.StuI.Xhol.SstI.Nsil.BclI

-w

1.3 kb _

0.7 kb --_

0 30 lb 2h4h 0

30'1 2h2 3h

Nothern blot analysis of total RNA (20ug) isolated from cells stimulated

These constructs provide

efficient tool for rapid analysis of post-transcriptional

regulation such as mRNA half-life determination and protein turnover analysis. They avoid the

of drugs such as actinomycinD and cycloheximide, which

induce artefactual results.

*To whom correspondence should be addressed REFERENCES

1. Marx, J.L. (1987) Science 237:854-856 2. Ruther, U. et al. (1985) EMBO J. 4:1775-1781 3. van Ooyen, A. et al. (1979) Science 206:337-344 4. Fort, P. et al. (1987) Nucl. Acid Res. 15:5657-5667

2874

2874 (¢) I R L Press