RevTet Gene Expression Systems

Gene expression systems are used by geneticists, molecular biologists, and researchers in industry to regulate the activity of genes introduced into their test organisms. Clontech Laboratoy Inc. markets a tetracycline dependant gene expression system which can regulate the activity of an inserted gene.

I do not have this system, and am thus unable to market it to you. If you would like to purchase this system or would like more technical information, go to the Clontech Catalog.

The RevTet-OnTM and RevTet-OffTM systems can be established in stable cell lines of E. coli. These systems use retroviruses to infect the host cells. The company states that the retrovirus will infect all dividing cells with up to100% efficiency. The gene expression is regulated at the transcription level. Upstream from the desired gene, the RevTet virus inserts a set of inhhibitors which can block transcription of the gene. The RevTet-Off system includes a regulatory element which binds to teracycline response element (TRE) and blocks transcription downstream. Bugs produce TRE in the presence of tetracycline and tetracycline doxycycline. Either antibiotic given dosewise will have a correlating effect on transcription. By controlling the ammount of tetracycline in the enviornment, the researcher can express the gene at different levels of activity.
The RevTet-On expression system is similar, but contains an inhibitor which binds to tetracycline doxycycline (DOX). As the concentration of DOX is increased, the more copies of the gene are transcribed and the gene is expressed to a greater degree. This double system then provides for both the positive and a negative regulation of genes as desired. 1

The Restriction maps for pRevTet-Off and pRevTet-On.

Follow these Clontech links to pRevTet-Off for the Vector Sequence, the Restriction Digest, the Vector Map & MCS, (in acrobat) and the Vector Sequence text.

Restriction map of pRevTet-Off. Unique restriction sites are in bold. 2 The Amp resistance in this plasmid allows for antibiotic selection.

Follow these Clontech links to pRevTet-On for the Vector Sequence, the Restriction Digest, the Vector Map & MCS, (in acrobat) and the Vector Sequence text.

Restriction map of pRevTet-On. Unique restriction sites are in bold. 3

Why Use this Gene Expression System?

    One good attribute of this system is that the system can be established in stable cells lines. E. coli with the desired gene and regulatory system can be maintained through many generations. This feature allows for a change of enviornments to be effected before a gene is turned on, or off. Also, the claimed up to100% transduction into dividing cells is an adequate rate for most research. The system's ability to regulate the rate of transcription is ideal for studies interested in concentrations of proteins in the cell. A gradient of gene product concentration is simply the result of scaled dosages of tetracycline.
    One possible use of the system would be to study the interaction of two proteins within a cell. One gene could be transduced into a cell without a regulatory system, and the other transduced with a Rev-Tet system. The restriction sites for this double transduction would have to be carefully chosen so as not to disrupt either plasmid. With the regulation of tetracycline, the regulated gene can be expressed at a range of activities and the resultant interaction between the two transfected genes can be studied. In this scenario, each gene should alternately be the unregulated and the regulated gene and controls of each gene, regulated and unregulated, and both genes unregulated should be run.
    This system works in vivo4, and this allows for delayed, pulsed, and otherwise regulated gene expression in an organism. This is a platform from which any number of experiments can be launched, particularliy in embryology and development in organisms resistant to tetracycline and which don't digest the antibiotic into components too quickly.
Two good thoughts that were realized to be impractible:
    With the first proposed experimantal procedure, what of regulating both genes with the tetracycline, or counter-regulating them with RevTet-Off and RevTet-On? This would be good experiment and control if it were feasible, but as the plasmids would be inserting with the same restriction sites, and you would be unable to detemine by reporters if both plasmids transfected successfully, these ideas will not work. For these, a second gene expression system with differing restriction sites and reporter system would be desired.

Special thanks to Kristi Dhoner at Clontech Laboratories Inc. for permission to cite their pages and to use technical graphics. 5

1 1998 Aug 4. CLONTECH Catalog:Retroviral Tet Expression Systems. <http://www.clontech.com/
clontech/Catalog/GeneExpression/RevTet.html> Accessed 1999 Feb 14.

2 1998 Jun 10. pRevTet-Off Vector Information. <http://www.clontech.com/clontech/Vectors/pRevTet-Off.html> Accessed 1999 Feb 14.

3 1998 Jun 10. pRevTet-On Vector Information. <http://www.clontech.com/clontech/Vectors/pRevTet-On.html> Accessed 1999 Feb 14.

4 1998 Aug 4. CLONTECH Catalog:Retroviral Tet Expression Systems. <http://www.clontech.com/
clontech/Catalog/GeneExpression/RevTet.html> Accessed 1999 Feb 14.

5 Dohner K. <kkdohner@clontech.com> 1999 Feb 11. RE: CLONTECH WWW Comments [Personal email]. Accessed 1999 Feb 14.