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Essential Components of the Tc-controlled Regulatory System

Regulator Plasmids.

The tTA-encoding vectors.

The basic plasmid (pUHD15-1) [1] encodes a CMV promoter/enhancer, the coding sequence for tTA and a SV40 polyadenylation site. An alternative plasmid, pUHG15-1 [10] contains the -globin intron/poly (A) site instead of the SV40 poly (A) site. This may be particularly useful when tTA expression is examined by RNA analysis. In our experience, the presence or absence of an intron does not result in different levels of tTA synthesis.

We recommend the hCMV promoter/enhancer for tTA expression since it has been successfully used in a number of cell lines. Depending on the specific application, particularly when tissue-specific expression in transgenic organisms is the goal, one has to consider more appropriate expression signals. The optimal promoter/enhancer can only be defined in the context of an experiment with respect to a particular cell line or organism.

We strongly recommend not to modify the tTA ORF! Several attempts (including our own) e. g. insertion of nuclear localization sequences have resulted in a hampered regulatory system.

The rtTA-encoding vectors.

Two versions of rtTA have been described (2). rtTA as encoded in pUHD17-1 or pUHG17-1 differs from tTA only by 4 amino acid exchanges, resulting in the reverse phenotype. Beside the respective nucleotide exchanges pUHD17-1 is identical to pUHD15-1 (see above). For analytical purpose the two plasmids can be distinguished by an additional HindIII site in the rtTA coding region. rtTA-nls as encoded by pUHD172-1neo contains an additional N-terminal nuclear localisation sequence (2). In addition, this plasmid contains a neomycin resistance cassette.

Response plasmids

The response plasmids such as pUHD10-3 (5) and its derivatives can be used for the tTA as well as for the rtTA system. They have a tTA-dependent promoter in front of a multiple cloning site or of a gene of interest. We strongly recommend to examine tTA or rtTA function with a tTA-controlled reporter unit. Our prefered reporter system is luciferase (pUHC13-3). If luciferase is not a suitable reporter, pUHG16-3 (10) encoding tTA-controlled -galactosidase can be used.

The Effector Substance.

We now use preferentially doxycycline hydrochloride (Dox-HCl, which, like tetracycline hydrochloride, is water soluble!) which functions with tTA and rtTA. We use a freshly made stock solution of Dox-HCl for 2 weeks (1 mg/ml; filter-sterilized and stored at 4oC in the dark). Alternatively, stock solutions can be frozen in small aliquots at -20oC, for long term storage. Dox-HCl in PBS forms a precipitate after a few days; medium supplemented with Dox-HCl should therefore not be stored for prolonged times. Tetracycline-HCl is only marginally functional in the rtTA system. The most potent rtTA effector so far identified by us is doxycycline-HCl. To feed doxycycline to transgenic animals, we routineously dissolve this substance at a final concentration of 200 g/ml in deionized water (water bottle should be light protected). The drinking water which should be renewed every 3 days contains 5 % succrose.

Culture media.

In our as well as in several other laboratories, it has been observed that highly regulatable cell lines suddenly do not express the gene of interest anymore when incubated in "Tc-free" medium. A thorough analysis of several of these incidents revealed that in each case a change in the batch of calf serum or fetal calf serum had occurred and we know today that commercial serum preparations can shut off tTA responsive promoters. We suspect that they contain Tc or one of its derivatives. We are in the process of identifying such tetracyclines in some batches of serum. A good way to examine the quality of sera in this respect is to grow and regulate the X1 cell line described in our 1992 PNAS-paper. This cell line will soon be available through the ECACC (European Collection of Cell Cultures, CAMR, Salisbury, Wiltshire SP4 0JG, UK, Tel. +44-1980-612512, Fax +44-1980-611315).

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