Transgenic Research

, Volume 21, Issue 2, pp 415–428 | Cite as

A doxycycline-inducible, tissue-specific aromatase-expressing transgenic mouse

  • Jenny D. Y. Chow
  • John T. Price
  • Margaret M. Bills
  • Evan R. Simpson
  • Wah Chin Boon
Technical Report


Aromatase converts androgens to estrogens and it is expressed in gonads and non-reproductive tissues (e.g. brain and adipose tissues). As circulating levels of estrogens in males are low, we hypothesize that local estrogen production is important for the regulation of physiological functions (e.g. metabolism) and pathological development (e.g. breast and prostate cancers) by acting in a paracrine and/or intracrine manner. We generated a tissue-specific doxycycline-inducible, aromatase transgenic mouse to test this hypothesis. The transgene construct (pTetOAROM) consists of a full-length human aromatase cDNA (hAROM) and a luciferase gene under the control of a bi-directional tetracycline-responsive promoter (pTetO), which is regulated by transactivators (rtTA or tTA) and doxycycline. Our in vitro studies using MBA-MB-231tet cells stably expressing rtTA, showed that doxycycline treatment induced transgene expression of hAROM transcripts by 17-fold (P = 0.01), aromatase activity by 26-fold, (P = 0.0008) and luciferase activity by 9.6-fold (P = 0.0006). Pronuclear microinjection of the transgene generated four pTetOAROM founder mice. A male founder was bred with a female mammary gland-specific rtTA mouse (MMTVrtTA) to produce MMTVrtTA-pTetOAROM double-transgenic mice. Upon doxycycline treatment via drinking water, human aromatase expression was detected by RT-PCR, specifically in mammary glands, salivary glands and seminal vesicles of double-stransgenic mice. Luciferase expression and activity was detected in these tissues by in vivo bioluminescence imaging, in vitro luciferase assay and RT-PCR. In summary, we generated a transgenic mouse model that expresses the human aromatase transgene in a temporal- and spatial-specific manner, which will be a useful model to study the physiological importance of local estrogen production.


Conditional transgenic Aromatase Luciferase Doxycycline Tet-ON MMTV 





Human aromatase gene/cDNA


Firefly luciferase cDNA


Mouse mammary tumour virus (promoter)


Tetracycline-responsive promoter


Reverse transactivation



This work was supported by Australian NH&MRC Project Grant, #494813 to WC Boon, #395525 R Douglas Wright Fellowship to JT Price and NHMRC Equipment Grant #467202 to JT Price and by # 338510 and Program Grant # 441100, as well as a Program Grant from the Victorian Breast Cancer Research Consortium Inc to ER Simpson.


  1. Ackerman GE, Smith ME et al (1981) Aromatization of androstenedione by human adipose tissue stromal cells in monolayer culture. J Clin Endocrinol Metab 53:412–417PubMedCrossRefGoogle Scholar
  2. Albanese C, Reutens AT et al (2000) Sustained mammary gland-directed, ponasterone A-inducible expression in transgenic mice. Faseb J 14(7):877–884PubMedGoogle Scholar
  3. Böcker R, Estler CJ et al (1981) Comparison of distribution of doxycycline in mice after oral and intravenous application measured by a high-performance liquid chromatographic method. Arzneimittelforschung 31(12):2116–2117PubMedGoogle Scholar
  4. Böcker R, Warnke L et al (1984) Blood and organ concentrations of tetracycline and doxycycline in female mice. Comparison to males. Arzneimittelforschung 34(4):446–448PubMedGoogle Scholar
  5. Boon WC, Diepstraten J et al (2005) Hippocampal NMDA receptor subunit expression and watermaze learning in estrogen deficient female mice. Mol Brain Res 140(1–2):127–132PubMedCrossRefGoogle Scholar
  6. Bulun SE, Sebastian S et al (2003) The human CYP19 (aromatase P450) gene: update on physiologic roles and genomic organization of promoters. J Steroid Biochem Mol Biol 86(3–5):219–224PubMedCrossRefGoogle Scholar
  7. Carani C, Qin K et al (1997) Effect of testosterone and estradiol in a man with aromatase deficiency. N Engl J Med 337(2):91–95PubMedCrossRefGoogle Scholar
  8. Cawthorne C, Swindell R et al (2007) Comparison of doxycycline delivery methods for Tet-inducible gene expression in a subcutaneous xenograft model. J Biomol Tech 18(2):120–123PubMedGoogle Scholar
  9. Chen D, Reierstad S et al (2009) Regulation of breast cancer-associated aromatase promoters. Cancer Lett 273(1):15–27PubMedCrossRefGoogle Scholar
  10. Chow JDY, Simpson ER et al (2009) Alternative 5′-untranslated first exons of the mouse Cyp19A1 (aromatase) gene. J Steroid Biochem Mol Biol 115(3–5):115–125PubMedCrossRefGoogle Scholar
  11. Connelly S (1999) Adenoviral vectors for liver-directed gene therapy. Curr Opin Mol Ther 1(5):565–572PubMedGoogle Scholar
  12. Demura M, Reierstad S et al (2008) Novel promoter I.8 and promoter usage in the CYP19 (Aromatase) gene. Reprod Sci 15(10):1044–1053PubMedCrossRefGoogle Scholar
  13. Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89(12):5547–5551PubMedCrossRefGoogle Scholar
  14. Gunther EJ, Belka GK et al (2002) A novel doxycycline-inducible system for the transgenic analysis of mammary gland biology. FASEB J 16(3):283–292PubMedCrossRefGoogle Scholar
  15. Harada N, Honda S (2005) Analysis of spatiotemporal regulation of aromatase in the brain using transgenic mice. J Steroid Biochem Mol Biol 95(1–5):49–55PubMedCrossRefGoogle Scholar
  16. Harada N, Utsumi T et al (1993) Tissue-specific expression of the human aromatase cytochrome P-450 gene by alternative use of multiple exons 1 and promoters, and switching of tissue-specific exons 1 in carcinogenesis. Proc Natl Acad Sci USA 90(23):11312–11316PubMedCrossRefGoogle Scholar
  17. Harada N, Wakatsuki T et al (2009) Functional analysis of neurosteroidal oestrogen using gene-disrupted and transgenic mice. J Neuroendocrinol 21(4):365–369PubMedCrossRefGoogle Scholar
  18. Hinshelwood MM, Mendelson CR (2001) Tissue-specific expression of the human CYP19 (aromatase) gene in ovary and adipose tissue of transgenic mice. J Steroid Biochem Mol Biol 79(1–5):193–201PubMedCrossRefGoogle Scholar
  19. Hinshelwood MM, Smith ME et al (2000) A 278 bp region just upstream of the human CYP19 (aromatase) gene mediates ovary-specific expression in transgenic mice. Endocrinology 141(6):2050–2053PubMedCrossRefGoogle Scholar
  20. Hruska KS, Tilli MT et al (2002) Conditional over-expression of estrogen receptor alpha in a transgenic mouse model. Transgenic Res 11(4):361–372PubMedCrossRefGoogle Scholar
  21. Kamat A, Graves KH et al (1999) A 500-bp region, approximately 40 kb upstream of the human CYP19 (aromatase) gene, mediates placenta-specific expression in transgenic mice. Proc Natl Acad Sci USA 96(8):4575–4580PubMedCrossRefGoogle Scholar
  22. Konopka W, Duniec K et al (2009) Tet system in the brain: transgenic rats and lentiviral vectors approach. Genesis 47(4):274–280PubMedCrossRefGoogle Scholar
  23. Labbe GG, Fromenty BB et al (1991) Effects of various tetracycline derivatives on in vitro and in vivo beta-oxidation of fatty acids, egress of triglycerides from the liver, accumulation of hepatic triglycerides, and mortality in mice. Biochem Pharmacol 41(4):638–641PubMedCrossRefGoogle Scholar
  24. Lephart ED, Simpson ER (1991) Assay of aromatase activity. Methods Enzymol 206:477–483Google Scholar
  25. Li X, Nokkala E et al (2001) Altered structure and function of reproductive organs in transgenic male mice overexpressing human aromatase. Endocrinology 142(6):2435–2442PubMedCrossRefGoogle Scholar
  26. Maffei L, Murata Y et al (2004) Dysmetabolic syndrome in a man with a novel mutation of the aromatase gene: effects of testosterone, alendronate, and estradiol treatment. J Clin Endocrinol Metab 89(1):61–70PubMedCrossRefGoogle Scholar
  27. Maffei L, Rochira V et al (2007) A novel compound heterozygous mutation of the aromatase gene in an adult man: reinforced evidence on the relationship between congenital oestrogen deficiency, adiposity and the metabolic syndrome. Clin Endocrinol (Oxf) 67(2):218–224CrossRefGoogle Scholar
  28. Mahendroo MS, Mendelson CR et al (1993) Tissue-specific and hormonally controlled alternative promoters regulate aromatase cytochrome P450 gene expression in human adipose tissue. J Biol Chem 268(26):19463–19470PubMedGoogle Scholar
  29. McNatty KP, Baird DT et al (1976) Concentration of oestrogens and androgens in human ovarian venous plasma and follicular fluid throughout the menstrual cycle. J Endocrinol 71(1):77–85PubMedCrossRefGoogle Scholar
  30. Morishima A, Grumbach MM et al (1995) Aromatase deficiency in male and female siblings caused by a novel mutation and the physiological role of estrogens. J Clin Endocrinol Metab 80(12):3689–3698PubMedCrossRefGoogle Scholar
  31. Robertson KM, O’Donnell L et al (1999) Impairment of spermatogenesis in mice lacking a functional aromatase (cyp 19) gene. PNAS 96(14):7986–7991PubMedCrossRefGoogle Scholar
  32. Ross SR, Hsu CL et al (1990) Negative regulation in correct tissue-specific expression of mouse mammary tumor virus in transgenic mice. Mol Cell Biol 10(11):5822–5829PubMedGoogle Scholar
  33. Sasano H, Nagura H et al (1994) Immunolocalization of aromatase and other steroidogenic enzymes in human breast disorders. Hum Pathol 25(5):530–535PubMedCrossRefGoogle Scholar
  34. Simpson ER (2003) Sources of estrogen and their importance. J Steroid Biochem Mol Biol 86(3–5):225–230PubMedCrossRefGoogle Scholar
  35. Simpson ER, Evans CT et al (1987) Sequencing of cDNA inserts encoding aromatase cytochrome P-450 (P-450AROM). Mol Cell Endocrinol 52(3):267–272 Google Scholar
  36. Sjögren K, Lagerquist M et al (2009) Elevated aromatase expression in osteoblasts leads to increased bone mass without systemic adverse effects. J Bone Miner Res 24(7):1263–1270PubMedCrossRefGoogle Scholar
  37. Tekmal RR, Ramachandra N et al (1996) Overexpression of int-5/aromatase in mammary glands of transgenic mice results in the induction of hyperplasia and nuclear abnormalities. Cancer Res 56(14):3180–3185PubMedGoogle Scholar
  38. Yamamoto N, Christenson LK et al (2002) Growth differentiation factor-9 inhibits 3′5′-adenosine monophosphate-stimulated steroidogenesis in human granulosa and theca cells. J Clin Endocrinol Metab 87(6):2849–2856PubMedCrossRefGoogle Scholar
  39. Yarranton GT (1992) Inducible vectors for expression in mammalian cells. Curr Opin Biotechnol 3(5):506–511PubMedCrossRefGoogle Scholar
  40. Zhang N, Weber A et al (2003) An inducible nitric oxide synthase-luciferase reporter system for in vivo testing of anti-inflammatory compounds in transgenic mice. J Immunol 170(12):6307–6319PubMedGoogle Scholar
  41. Zhou J, Suzuki T et al (2005) Interactions between prostaglandin E2, liver receptor homologue-1, and aromatase in breast cancer. Cancer Res 65(2):657–663PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Jenny D. Y. Chow
    • 1
    • 2
  • John T. Price
    • 3
  • Margaret M. Bills
    • 3
  • Evan R. Simpson
    • 1
    • 3
  • Wah Chin Boon
    • 1
    • 2
    • 4
  1. 1.Prince Henry’s InstituteClaytonAustralia
  2. 2.Department of Anatomy and Developmental BiologyMonash UniversityClaytonAustralia
  3. 3.Department Biochemistry and Molecular BiologyMonash UniversityClaytonAustralia
  4. 4.Florey Neuroscience InstitutesThe University of MelbourneParkvilleAustralia

Personalised recommendations