Shwu-Yuan Wu and Cheng-

A tetracycline-regulated expression system is combined with the FLAG-epitope tagging method for conditional expression of potentially toxic proteins in mammalian cells. This strategy allows a controlled expression of exogenous gene products and also provides a unique way of protein purification. Two mammalian expression plasmids containing the FLAG sequence and flanking multiple cloning sites were created for conditional protein expression. The cDNAs encoding human basal transcription factors TBP, TAFII55 and the p62 subunit of TFIIH were individually cloned into these vectors and introduced into a HeLa-derived cell line that constitutively expresses a tetracycline-regulated transactivator (tTA). The established clonal human cell lines express FLAG-tagged basal transcription factors in a manner modulated by the amount of tetracycline in the growth medium. In the absence of tetracycline, tTA binds to the DNA recognition sites of the expression plasmid and induces the expression of tagged proteins. When tetracycline is added back to the growth medium, the induced protein starts to decay. This provides us with an estimation of the in vivo half-lives of TBP and TAFII55, which were assessed to be less than 20 and 6 hours, respectively, in HeLa cells. The level of induced proteins in the absence of tetracycline could be further enhanced by including the antibiotic G418 to presumably boost the production of tTA which in turn activates the expression of tagged proteins. INTRODUCTION The biological function of proteins is often investigated by transient overexpression of a particular gene product in cultured cells. Transient transfection offers a quick and simple assay to evaluate the potential activity of proteins, such as transcription factors and cellcycle regulatory proteins. However, due to the variable proportion of cells taking up the DNA and to variations in culture conditions, transient transfection is sometimes difficult to repeat, and frequently cannot provide enough transfected cells for biochemical analysis. In our experience, successful purification of a multisubunit protein complex is unlikely to be achieved by simply overexpressing a single protein subunit by transient transfection. This problem is further complicated by the stoichiometric ratio among all other subunits in a multiprotein complex. To overcome these difficulties, we and others have used retrovirus-mediated gene transfer techniques to establish stable cell lines that constitutively express epitope-tagged proteins for purification of multiprotein complexes, such as the basal transcription factor TFIID (1,13). The introduced epitope tag also provides a convenient site for affinity purification by monoclonal antibodyconjugated beads. This purification strategy has been successfully applied to many other protein complexes (1; S.Y. Wu and C.-M. Chiang, unpublished data). Nevertheless, attempts to establish stable cell lines that express other transcription factors by a retroviral approach have failed, partly due to the potential cytotoxicity of these proteins. Conditional expression of a foreign gene product is invaluable in assessing the biological function of a protein during specific stages of development, cellular growth and division cycles. Conversely, inhibition of endogenous protein expression can also reveal the effect(s) of the protein on cellular activities. However, it has been difficult to produce effective regulated expression for studies in vertebrate cells. Recently, a tetracycline-regulated expression system (9) has been successfully applied to mammalian cells for controlled expression of an ectopic gene product. This inducible system relies on a bacterial tetracycline repressor (TcR) that binds to DNA recognition sites only in the absence of tetracycline. In the presence of the antibiotic, ligand-induced conformational change of the TcR DNA-binding domain prevents it from binding to the cognate DNA sequence. This “on” and “off” regulation offers a precise control for protein expression in mammalian cells because tetracycline does not change the overall metabolism in mammalian cells, as do other inducers such as heat shock, heavy metals and hormones (8). This approach has also been used to create transgenic mice for conditional expression of specific gene products (7). For this system to work in mammalian cells, the DNAbinding domain of TcR was linked to the acidic activation domain of VP16. This tetracycline-dependent transactivator (tTA), i.e., TcR-VP16 fusion protein, acts as a transcriptional activator when it binds to specific DNA sites located upstream of eukaryotic promoters used to drive the expression of inserted genes. A HeLa-derived cell line (HtTA1) that constitutively expresses tTA was established for induced expression of foreign gene products (9). Overexpression of a crucial tran718 BioTechniques Vol. 21, No. 4 (1996) Establishment of Stable Cell Lines Expressing Potentially Toxic Proteins by TetracyclineRegulated and Epitope-Tagging Methods BioTechniques 21:718-725 (October 1996) scription factor in cells may disturb cellular metabolism, resulting in cell death. This might be due to cytotoxicity exerted by unregulated expression of ectopic gene products. Since we could not establish stable cell lines expressing several epitope-tagged transcription factors using retrovirus-mediated gene transfer, we have resorted to the tetracycline-regulated expression system that can turn gene expression “on” or “off” depending on the availability of tetracycline (9). In this report, we describe an adaptation of the tetracyclineregulated expression system, in combination with FLAG epitope-tagging (2), to establish stable cell lines that conditionally express potentially toxic proteins which can be easily purified for biochemical analysis. MATERIALS AND METHODS Construction of TetracyclineRegulated Plasmids Expressing FLAG-Tagged Basal Transcription Factors Two oligonucleotides, 5′ CATGGACTACAAAGACGATGACGATAAACA-3′ and 5′-TATGTTTATCGTCATCGTCTTTGTAGTC-3′, were individually synthesized and annealed to form a double-stranded oligonucleotide. This double-stranded oligonucleotide containing the FLAG epitope sequence with flanking NcoI and NdeI sites was used to replace the original FLAG sequence that also contains a kinase phosphorylation site in both pFLAG(S)-7 (2) and pFLAG(AS)-7 (1) to create, respectively, pFLAGo(S)-7 and pFLAGo (AS)-7 (Figure 1A). All these FLAGcontaining cloning plasmids have a Kozak consensus sequence (10) inserted between the BglII and NcoI sites to facilitate expression in mammalian cells. The EcoRI-BamHI fragment of pFLAGo(S)-7 was then cloned into pUHD10-3 (11) at the corresponding sites to generate pTetCMV-Fo(S) (Figure 1B). Similarly, the ClaI-XbaI fragment of pFLAGo(AS)-7 was cloned into pUHD10-3 between the EcoRI and XbaI sites after Klenow filling-in at the ClaI and EcoRI sites (which regenerates the EcoRI site) to make pTetCMVFo(AS) (Figure 1B). For conditional expression of FLAG-tagged human TBP, the NdeI-BamHI fragment of TBP was transferred from pF:TBP(S)-7 (2) to pTetCMV-Fo(S) to create pTetCMVFo:hTBP. To make tetracycline-regulated human TAFII55 expression plasmids, the NdeI-SacI fragment of pF:55(ORF)-11d (3) and the SacIEcoRI fragment of 7-21 (3) were cloned into pFLAGo(AS)-7 between the NdeI and EcoRI sites to create pFo:55-7, which contains TAFII55 cDNA from the initiation codon to the end of the 3′ untranslated region. The NcoI and partial XbaI digestion fragments of pFo:55-7 were then cloned into pTetCMV-Fo(AS) between the NcoI and XbaI sites to generate pTetCMV-F:hTAFII55 and pTetCMVF:hTAFII55/A. The former plasmid has a short 3′ untranslated region ending at the first XbaI site of TAFII55, while the latter plasmid contains the entire 3′ untranslated region, including the poly(A) signal, of TAFII55 and terminates at the XbaI site of the vector. The p62 cDNA of human TFIIH was isolated from pET11a/p62 (6) as N-terminal NdeI (introduced at the first initiation codon)-EcoRV and C-terminal EcoRVBamHI fragments, and cloned into pFLAG(S)-7 and pF:TBP-11d (2), after removing the TBP insert, between the NdeI and BamHI sites to generate pF:62(H)-7 and pF:62(H)-11d, respectively. The entire FLAG-tagged p62 cDNA was then cleaved from pF: 62(H)-7 by BamHI and partial EcoRI digestion, and cloned into pUHD10-3 between the EcoRI and BamHI sites to create pTetCMV-F:62(H) for conditional expression of the p62 subunit of human TFIIH in mammalian cells. Establishment of TetracyclineRegulated Clonal Human Cell Lines To establish stable cell lines that conditionally express epitope-tagged TBP, TAFII55 and p62, we cotransfected 10 μg of each PvuI-linearized pTetCMV-Fo:hTBP, pTetCMV-F:hTAFII55, pTetCMV-F:hTAFII55/A or pTetCMV-F:62(H) with 50 μg of sheared calf thymus DNA and with 0.5 μg of SacI-linearized pREP4 (Invitrogen, San Diego, CA, USA) that contains the hygromycin-resistant gene into HtTA-1 (9), a HeLa-derived cell line that constitutively expresses a tetracycline-controlled tetR-VP16 transactivator (tTA). Transfection was performed by electroporation (Bio-Rad, Hercules, CA, USA) at 960 μF and 200 V as previously described (4). After electroporation, the cells were left at room temperature for 10 min and then transferred to a 15mL centrifuge tube containing 10 mL growth medium (Dulbecco’s modified Eagle medium [DMEM] plus 10% fetal bovine serum [FBS]). Following spinning at 1000 rpm using a Sorvall® RC3B Plus centrifuge with an H6000A rotor (Du Pont, Wilmington, DE, USA) for 5 min to remove debris, the cells were plated onto five 100-mm plates and left in a 37°C, 5% CO2 incubator for two days before initiating drug selection (400 μg/mL of G418, 200 μg/mL of hygromycin B and 2 μg/mL of tetracycline). G418 sulfate (Geneticin®; Life Technologies, Gaithersburg, MD, USA) and tetracycline (Sigma Chemical, St. Louis, MO, USA) were dissolved in water as 1 mg/mL stock solution, whereas hygromycin B (Boehringer Mannheim, Indianapolis, IN, USA) was purchased as 20 mg/mL solution. These antibiotics were added to the growth medium before use. The medium was changed every 3 or 4 days and selection was continued for approximately 3 weeks until drug-resistant colonies were clearly visible. Twelve colonies were then picked up and expanded into cell lines. To identify positive clones responding to tetracycline, cells were plated onto two 60mm dishes in selection medium, with or without tetracycline, for 48 to 72 h. Subconfluent cells were then lysed in 250 μL of sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and used for Western blotting.