Introduction

The ability to introduce and express exogenous DNA in a controlled manner in mammalian cells plays an important role in the study of biological phenomena as well as in enabling the generation of new tools for therapeutic ends, such as gene therapy (1,2). Many approaches have been developed to exactly control the spatial and temporal expression of exogenous genes, typically applying the regulation at the transcriptional level and thereby controlling messenger RNA (mRNA) copy number and subsequently protein level (2), but other, more complex regulation systems have been implemented (3,4,5,6,7).

One of the best transcriptional regulatory systems, often termed the tet inducible system, consists of two components, the tetracycline transcription activator protein (tTA) and a DNA operator sequence, tetO (8,9,10). The transactivator is a chimeric protein consisting of the VP16 transcription activation domain and the Escherichia coli tet-repressor DNA binding domain. In the Tet-Off version, in the absence of the tetracycline derivative doxycycline (Dox), the tet repressor binds to the tetO sequence, bringing the VP16 subunit close enough to DNA to initiate transcription of the regulated gene. Dox addition induces a conformational change in the tet repressor, which prevents the association of the tet repressor with the DNA; thus, when Dox is added, transcriptional activity (mediated by VP16) is repressed. The Tet-Off system has been used extensively in vitro and in vivo for protein expression purposes, yet some shortcomings still exist that limit its utility: most notably, detectable background expression occurs even in the continual presence of Dox. Furthermore, the system is not protected from Dox fluctuations in the sense that a small decrease in the concentration of Dox would render the system active, the extent of activity depending on the concentration of Dox.

Another synthetic regulatory system is based on the ability to control protein stability (11,12) via small molecule intervention. FKBP12-rapamycin binding domain of mTOR (FRB) is a highly unstable 89–amino acid destruction domain derived from the mTOR protein (13,14) that is rapidly degraded in cells. When expressed as a fusion with another protein, this instability is conferred on the fused protein in a cis-dominant manner, leading to rapid turnover of the fusion protein in mammalian cells. Rapamycin (Rap) is a natural antifungal, cell-permeable compound also capable of serving as an immunosuppressant by inhibiting the activity of the mTOR pathway (13,14). Notably, FRB and FRB-fusion proteins are stabilized when Rap is added to the cellular media (15), but as Rap is an mTOR pathway inhibitor, its use in cellular systems may be challenging. In response to this potential complication, other inducibly stabilized domains have been developed that are stabilized by what so far have been shown to be substances that do not affect cellular activity (15,16). For instance, FRB variants (i.e., FRB*; see Reference 15) can respond to a rapamycin substitute, and the FKBP* inducibly stabilized domain is responsive to the biologically inert synthetic molecule Shld-1 (16).

We investigated how regulating protein stability may be introduced into the tTA system and define two modes of combining regulatory modules (17). In the first, the parallel regulation approach, destabilization is applied to the control protein (tTA). In the second, the serial regulation approach, the destabilization is applied to the protein already regulated on the transcriptional level. We show that each strategy has very distinct advantages and applications.

Materials and Methods

Vectors

A sequence coding for transcription initiation followed by the FRB* coding sequence was cloned into the MFG-I-CD8 vector, a constitutive retroviral expression vector for expressing a gene of interest followed by an internal ribosomal entry site (IRES) element, and CD8 as a marker. The resulting construct, MFG-FRB*, contains FRB* inserted within the MluI and SalI restriction sites. We used primers for the N and C termini of tTA in the Retrotet RTAb(−) vector to PCR-amplify a tTA coding sequence containing no start codon. This tTA (lacking the first ATG) cDNA was then cloned in-frame into MFG-FRB* using SalI and NotI restriction sites. The resultant vector expresses an FRB*-tTA fusion protein (termed F*tTA hereafter) and an IRES-expressed cell surface marker, CD8. The same procedure was carried out to produce K*tTA, which is a fusion of FKBP* and tTA. Unless mentioned otherwise, the fusion proteins are separated only by the two amino acids coded by the SalI site (GTCGAC). The MFG-I-CD8 and Retrotet vectors were gifts from Helen Blau (Stanford University, Palo Alto, CA, USA), and FKBP* cDNA was provided by Tom Wandless (Stanford University).