Retroviral vectors for establishing tetracycline-regulated gene expression in an otherwise recalcitrant cell line

BMC Molecular Biology

Alan Ashworth²  Tariq Enver³  Paraic A Kenny¹ 

[1] Current address: Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720 USA;The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, United Kingdom;Section of Gene Function and Regulation, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, United Kingdom

关键词: transgenics;    retroviral vectors;    inducible expression;    doxycycline;    tetracycline;   

Others  :  1142241
DOI  :  10.1186/1471-2199-3-13

 received in 2002-06-19, accepted in 2002-09-03,  发布年份 2002

【 摘 要 】

Background

Tetracycline-regulated systems have been used to control the expression of heterologous genes in such diverse organisms as yeast, plants, flies and mice. Adaptation of this prokaryotic regulatory system avoids many of the problems inherent in other inducible systems. There have, however, been many reports of difficulties in establishing functioning stable cell lines due to the cytotoxic effects of expressing high levels of the tetracycline transactivator, tTA, from a strong viral promoter.

Results

Here we report the successful incorporation of tetracycline-mediated gene expression in a mouse mammary epithelial cell line, HC11, in which conventional approaches failed. We generated retroviruses in which tTA expression was controlled by one of three promoters: a synthetic tetracycline responsive promoter (TRE), the elongation factor 1-alpha promoter (EF1α) or the phosphoglycerate kinase-1 promoter (PGK), and compared the resulting cell lines to one generated using a cytomegalovirus immediate early gene promoter (CMV). In contrast to cells produced using the CMV and PGK promoters, those produced using the EF1α and TRE promoters expressed high levels of β-galactosidase in a tetracycline-dependent manner.

Conclusions

These novel retroviral vectors performed better than the commercially available system and may have a more general utility in similarly recalcitrant cell lines.

【 授权许可】

   
2002 Kenny et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

【 图 表 】

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【 参考文献 】

• [1]Kay RJ, Boissy RJ, Russnak RH, Candido EP: Efficient transcription of a Caenorhabditis elegans heat shock gene pair in mouse fibroblasts is dependent on multiple promoter elements which can function bidirectionally. Mol Cell Biol 1986, 6:3134-3143.
• [2]Kothary R, Clapoff S, Darling S, Perry MD, Moran LA, Rossant J: Inducible expression of an hsp68-lacZ hybrid gene in transgenic mice. Development 1989, 105:707-714.
• [3]Schweinfest CW, Jorcyk CL, Fujiwara S, Papas TS: A heat-shock-inducible eukaryotic expression vector. Gene 1988, 71:207-210.
• [4]Mayo KE, Warren R, Palmiter RD: The mouse metallothionein-I gene is transcriptionally regulated by cadmium following transfection into human or mouse cells. Cell 1982, 29:99-108.
• [5]Huang AL, Ostrowski MC, Berard D, Hager GL: Glucocorticoid regulation of the Ha-MuSV p21 gene conferred by sequences from mouse mammary tumor virus. Cell 1981, 27:245-255.
• [6]Webster N, Jin JR, Green S, Hollis M, Chambon P: The yeast UASG is a transcriptional enhancer in human HeLa cells in the presence of the GAL4 trans-activator. Cell 1988, 52:169-178.
• [7]Wang Y, O'Malley BW Jr, Tsai SY, O'Malley BW: A regulatory system for use in gene transfer. Proc Natl Acad Sci U S A 1994, 91:8180-8184.
• [8]Campbell SJ, Carlotti F, Hall PA, Clark AJ, Wolf CR: Regulation of the CYP1A1 promoter in transgenic mice: an exquisitely sensitive on-off system for cell specific gene regulation. J Cell Sci 1996, 109:2619-2625.
• [9]No D, Yao TP, Evans RM: Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc Natl Acad Sci U S A 1996, 93:3346-3351.
• [10]Hu MC, Davidson N: The inducible lac operator-repressor system is functional in mammalian cells. Cell 1987, 48:555-566.
• [11]Labow MA, Baim SB, Shenk T, Levine AJ: Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells. Mol Cell Biol 1990, 10:3343-3356.
• [12]Gossen M, Bujard H: Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A 1992, 89:5547-5551.
• [13]Bertrand KP, Postle K, Wray LV Jr, Reznikoff WS: Overlapping divergent promoters control expression of Tn10 tetracycline resistance. Gene 1983, 23:149-156.
• [14]Hinrichs W, Kisker C, Duvel M, Muller A, Tovar K, Hillen W, Saenger W: Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance. Science 1994, 264:418-420.
• [15]Gari E, Piedrafita L, Aldea M, Herrero E: A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast 1997, 13:837-848.
• [16]Weinmann P, Gossen M, Hillen W, Bujard H, Gatz C: A chimeric transactivator allows tetracycline-responsive gene expression in whole plants. Plant J 1994, 5:559-569.
• [17]Bello B, Resendez-Perez D, Gehring WJ: Spatial and temporal targeting of gene expression in Drosophila by means of a tetracycline-dependent transactivator system. Development 1998, 125:2193-2202.
• [18]Furth PA, St Onge L, Boger H, Gruss P, Gossen M, Kistner A, Bujard H, Hennighausen L: Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc Natl Acad Sci U S A 1994, 91:9302-9306.
• [19]Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H: Transcriptional activation by tetracyclines in mammalian cells. Science 1995, 268:1766-1769.
• [20]Gallia GL, Khalili K: Evaluation of an autoregulatory tetracycline regulated system. Oncogene 1998, 16:1879-1884.
• [21]Gopalkrishnan RV, Christiansen KA, Goldstein NI, DePinho RA, Fisher PB: Use of the human EF-1alpha promoter for expression can significantly increase success in establishing stable cell lines with consistent expression: a study using the tetracycline-inducible system in human cancer cells. Nucleic Acids Res 1999, 27:4775-4782.
• [22]Colosimo A, Goncz KK, Holmes AR, Kunzelmann K, Novelli G, Malone RW, Bennett MJ, Gruenert DC: Transfer and expression of foreign genes in mammalian cells. Biotechniques 2000, 29:314-324.
• [23]Ball RK, Friis RR, Schoenenberger CA, Doppler W, Groner B: Prolactin regulation of beta-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line. EMBO J 1988, 7:2089-2095.
• [24]Gill G, Ptashne M: Negative effect of the transcriptional activator GAL4. Nature 1988, 334:721-724.
• [25]Flint J, Shenk T: Viral transactivating proteins. Annu Rev Genet 1997, 31:177-212.
• [26]Baron U, Gossen M, Bujard H: Tetracycline-controlled transcription in eukaryotes: novel transactivators with graded transactivation potential. Nucleic Acids Res 1997, 25:2723-2729.
• [27]Urlinger S, Helbl V, Guthmann J, Pook E, Grimm S, Hillen W: The p65 domain from NF-kappaB is an efficient human activator in the tetracycline-regulatable gene expression system. Gene 2000, 247:103-110.
• [28]Go WY, Ho SN: Optimization and direct comparison of the dimerizer and reverse tet transcriptional control systems. J Gene Med 2002, 4:258-270.
• [29]Akagi K, Kanai M, Saya H, Kozu T, Berns A: A novel tetracycline-dependent transactivator with E2F4 transcriptional activation domain. Nucleic Acids Res 2001, 29:E23.
• [30]Pear WS, Nolan GP, Scott ML, Baltimore D: Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A 1993, 90:8392-8396.
• [31]Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227:680-685.