The C127 and C57 mouse cell lines were transfected with two Tc-regulatable plasmids, pG-BON and pDYAL-BON (Fig. ). Both plasmids carried all components of the Tc-regulatable system within a cassette controlled by a bi-directional TetP promoter. This promoter arrangement consists of the tetracycline-responsive element comprising seven copies of the 42-bp tet operator sequence. This element is positioned between two minimal CMV promoters that lack the enhancer that is part of the complete CMV promoter. The promoter is minimally expressed in the absence of binding of rtTA to the tet operator sequences. The promoter controls the luciferase gene and the rtTA reverse tetracycline transcriptional activator gene.
Figure 1 Tetracycline-inducible vectors. A tetracycline regulatable bidirectional cassette is carried by (A) a conventional plasmid, pG-BON and (B) an extrachromosomally retained, autonomously replicating EBV/human ori vector, pDYAL-BON. Amp r, ampicillin (more ...)
The pG-BON plasmid lacks the ability to replicate extrachromosomally, whereas pDYAL-BON can replicate extrachromosomally and is retained long-term in mammalian cells. pDYAL-BON contains the origin of replication, oriP
, from EBV, in which the dyad symmetry element needed for replication has been replaced with a 16.2 kb fragment of human alphoid DNA derived from the centromere of chromosome 17. This fragment confers the ability to replicate once per cell cycle in a variety of mammalian cells, including human, monkey and rodent cells (5
Selection for the neomycin or hygromycin resistance markers on the plasmids was carried out for 3 weeks. Populations of resistant cells were expected to consist of randomly integrated plasmid DNA in the case of pG-BON and extrachromosomally replicating plasmid in the case of pDYAL-BON. To confirm this expectation, plasmid DNA was purified from the cell populations by performing Hirt extraction (7
) and showing, in the case of pDYAL-BON-transfected mouse cells, the presence of plasmid DNA, all of which was Dpn
I-resistant. In the case of pG-BON-transfected mouse cells, no plasmid DNA was detected. The former result is consistent with the presence of extrachromosomal plasmid DNA that has undergone at least two rounds of replication in the mammalian cells. In practice, extrachromosomal plasmid DNA is present at this time after transfection only if the DNA has the ability to be retained and replicated in mammalian cells (8
The populations of resistant cells were used for experiments demonstrating doxycycline induction. Dox was added to culture medium at concentrations of 1 and 5 µg/ml, and luciferase induction was measured 24, 48 and 72 h later. Luciferase was maximally expressed in C127 and C57 cells within 48 h of doxycycline induction. Luciferase values are presented in Table and graphed in Figure . Measurements were also taken at 72 h, with the result that luciferase levels were stable, but not higher than at the earlier time points. It is typical of Tc-controlled gene expression systems to respond with similar kinetics. However, considering that expression of luciferase in our system is dependent on the prior production of rtTA through a positive feedback mechanism, reaching near maximum levels of luciferase by 24 h was a particularly favorable feature of this system.
Luciferase induction measurements
Doxycyline-mediated luciferase induction. The data in Table 1 are graphed here. Luciferase levels were evaluated 24 and 48 h after introduction of Dox. Experiments were performed in (A) C127 and (B) C57 mouse cell lines.
The bi-directional tetP promoter cassette was able to mediate Dox-controlled gene expression whether the regulating elements were carried on the conventional pG-BON plasmid or on the EBV/human ori pDYAL-BON vector. However, the EBV/human ori vector was consistently more effective. When the bi-directional unit was introduced into C57 mouse cells on the EBV/human ori vector, it was possible to induce luciferase expression >400-fold within 24 h of being exposed to 1 µg/ml Dox. In contrast, mouse cells transfected with the conventional pG-BON plasmid bearing the same bi-directional cassette induced expression of luciferase at a much lower level, in the range of 10–50-fold. The difference probably reflects the consistent gene expression observed on the extrachromosomal vector, as opposed to the randomly integrated vector, in which many of the integration sites may not induce well due to position effects.
The extrachromosomally replicating EBV/human ori
pDYAL-BON vector is generally present in multiple copies, ~1–100 per cell. The copy number tends to decrease slowly over time, even when selection is maintained (9
). Using the same cell lines that were assayed above at 3 weeks, we also induced luciferase expression with Dox at 1 month and obtained essentially identical results. For more prolonged studies, freezing of the transfected cells during the first month and subsequent thawing before use is recommended. We tested the freezing and thawing protocol on these transfected cells, frozen ~1 month after transfection. After thawing of the cells, Dox-induced luciferase expression had the same characteristics as that observed for the cells before freezing.
There are two advantages to including the Tc-controlled regulatable cassette on an EBV/human ori
vector. First, all the components involved in controlled regulation remain extrachromosomal. Gene expression from such extrachromosomal vectors has proven to be stable and consistent (9
), independent of the position effects and silencing that are routinely observed with chromosomally integrated transgenes. Therefore, each cell receiving the vector will generate approximately the same level of stable gene expression. It is not necessary to isolate clones, nor to screen such clones for optimal activity. This feature saves months of time and labor.
By using an EBV/human ori
vector instead of a conventional EBV vector, we gain the broad host range uniquely displayed by these vectors. Inclusion of a human genomic fragment to mediate replication in place of the dyad symmetry element present in the EBV oriP
viral origin frees the replication origin from the species specificity typical of viral origins of replication (10
). We have demonstrated replication function and long-term retention of such vectors in human, monkey, mouse and hamster cells (6
). No restrictions on vector replication have been observed, so it is likely that the vectors will also replicate in many other mammalian cell types.
A previous problem with inclusion of both the transactivator and the target gene on a multicopy extrachromosomal vector was an elevated supply of the transactivator, resulting in a poor induction ratio. To correct this problem, we created a situation in which the rtTA gene was highly expressed only when induced gene expression was desired, rather than constitutively expressing the transactivator. By placing the rtTA
gene under control of its own gene product, presence of the inducer is required to produce a significant amount of rtTA. In this way, the induction signal is amplified upon provision of the inducer, leading to a rapid and dramatic induction of gene expression of the regulated gene of interest. A similar idea has been applied previously in the context of a retroviral system (12
), but here the simplicity and convenience of a plasmid system is preserved.
This system should prove useful for rapidly creating mammalian cell lines carrying an inducible gene that is under tight control. This feature is especially valuable when creation of a large panel of inducible cell lines is required.