Fluorescent biosensors offer a powerful tool for tracking and quantifying protein activity in living systems with high temporospatial resolution. However, the expression of genetically encoded fluorescent proteins can interfere with endogenous signaling pathways, potentially leading to developmental and physiological abnormalities. The EKAREV-NLS mouse model, which carries a FRET-based biosensor for monitoring extracellular signal-regulated kinase (ERK) activity, has been widely utilized both in vivo and in vitro across various cell types and organs. In this study, we report a significant defect in mammary epithelial development in EKAREV-NLS C57BL/6J female mice. Our findings reveal that these mice exhibit severely impaired mammary epithelial outgrowth, linked to systemic defects including disrupted estrous cycling, impaired ovarian follicle maturation, anovulation, and reduced reproductive fitness. Notably, estrogen supplementation was sufficient to enhance mammary epithelial growth in the EKAREV-NLS C57BL/6J females. Furthermore, outcrossing to the ICR genetic background fully restored normal mammary epithelial outgrowth, indicating that the observed phenotype is dependent on genetic background. We also confirmed the functional performance of the biosensor in hormone-supplemented and outcrossed tissues through time-lapse imaging of primary mammary epithelial cells. Our results underscore the critical need for thorough characterization of biosensor-carrying models before their application in specific research contexts. Additionally, this work highlights the influence of hormonal and genetic factors on mammary gland development and emphasizes the importance of careful consideration when selecting biosensor strains for mammary studies.

Department of Histology and Embryology Faculty of Medicine Masaryk University Brno Czech Republic

Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Tao W, Evans B-G, Yao J, Cooper S, Cornetta K, Ballas CB, et al. Enhanced green fluorescent protein is a nearly ideal long-term expression tracer for hematopoietic stem cells, whereas DsRed-express fluorescent protein is not. Stem Cells. 2007;25:670–8. PubMed

Klein RL, Dayton RD, Leidenheimer NJ, Jansen K, Golde TE, Zweig RM. Efficient neuronal gene transfer with AAV8 leads to neurotoxic levels of tau or green fluorescent proteins. Mol Ther. 2006;13:517–27. PubMed PMC

Taghizadeh RR, Sherley JL. CFP and YFP, but not GFP, provide stable fluorescent marking of rat hepatic adult stem cells. J Biomed Biotechnol. 2008;2008:453590. PubMed PMC

Liu HS, Jan MS, Chou CK, Chen PH, Ke NJ. Is green fluorescent protein toxic to the living cells? Biochem Biophys Res Commun. 1999;260:712–7. PubMed

Ganini D, Leinisch F, Kumar A, Jiang J, Tokar EJ, Malone CC, et al. Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells. Redox Biol. 2017;12:462–8. PubMed PMC

Goto H, Yang B, Petersen D, Pepper KA, Alfaro PA, Kohn DB, et al. Transduction of green fluorescent protein increased oxidative stress and enhanced sensitivity to cytotoxic drugs in neuroblastoma cell lines. Mol Cancer Ther. 2003;2:911–7. PubMed

Coumans JVF, Gau D, Poljak A, Wasinger V, Roy P, Moens P. Green fluorescent protein expression triggers proteome changes in breast cancer cells. Exp Cell Res. 2014;320:33–45. PubMed PMC

Comley LH, Wishart TM, Baxter B, Murray LM, Nimmo A, Thomson D, et al. Induction of cell stress in neurons from transgenic mice expressing yellow fluorescent protein: implications for neurodegeneration research. PLoS ONE. 2011;6:e17639. PubMed PMC

Ansari AM, Ahmed AK, Matsangos AE, Lay F, Born LJ, Marti G, et al. Cellular GFP toxicity and immunogenicity: potential confounders in in vivo cell tracking experiments. Stem Cell Rev Rep. 2016;12:553–9. PubMed PMC

Huang WY, Aramburu J, Douglas PS, Izumo S. Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy. Nat Med. 2000;6:482–3. PubMed

Gasterstädt I, Jack A, Stahlhut T, Rennau L-M, Gonda S, Wahle P. Genetically encoded calcium indicators can impair Dendrite growth of cortical neurons. Front Cell Neurosci. 2020;14:570596. PubMed PMC

Kamioka Y, Sumiyama K, Mizuno R, Sakai Y, Hirata E, Kiyokawa E, et al. Live imaging of protein kinase activities in transgenic mice expressing FRET biosensors. Cell Struct Funct. 2012;37:65–73. PubMed

Harvey CD, Ehrhardt AG, Cellurale C, Zhong H, Yasuda R, Davis RJ, et al. A genetically encoded fluorescent sensor of ERK activity. Proc Natl Acad Sci U S A. 2008;105:19264–9. PubMed PMC

Komatsu N, Aoki K, Yamada M, Yukinaga H, Fujita Y, Kamioka Y, et al. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol Biol Cell. 2011;22:4647–56. PubMed PMC

Mizuno R, Kamioka Y, Kabashima K, Imajo M, Sumiyama K, Nakasho E, et al. In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines. J Exp Med. 2014;211:1123–36. PubMed PMC

Muta Y, Fujita Y, Sumiyama K, Sakurai A, Taketo MM, Chiba T et al. Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine. Nat Commun. 2018;9. PubMed PMC

Hiratsuka T, Fujita Y, Naoki H, Aoki K, Kamioka Y, Matsuda M. Intercellular propagation of extracellular signal-regulated kinase activation revealed by in vivo imaging of mouse skin. Elife. 2015;4:e05178. PubMed PMC

Aoki K, Kondo Y, Naoki H, Hiratsuka T, Itoh RE, Matsuda M. Propagating Wave of ERK Activation orients collective cell Migration. Dev Cell. 2017;43:305–e3175. PubMed

Kumagai Y, Naoki H, Nakasyo E, Kamioka Y, Kiyokawa E, Matsuda M. Heterogeneity in ERK activity as visualized by in vivo FRET imaging of mammary tumor cells developed in MMTV-Neu mice. Oncogene 2015. 2014;34:8. PubMed

Richardson FL. The relative growth of the mammary glands in normal, ovariectomized and adrenalectomized female mice. Anat Rec. 1955;123:279–89. PubMed

Haslam SZ. The ontogeny of mouse mammary gland responsiveness to ovarian steroid hormones. Endocrinology. 1989;125:2766–72. PubMed

Crabtree JS, Zhang X, Peano BJ, Zhang Z, Winneker RC, Harris HA. Development of a mouse model of mammary gland versus uterus tissue selectivity using estrogen- and progesterone-regulated gene markers. J Steroid Biochem Mol Biol. 2006;101:11–21. PubMed

Gordon MN, Osterburg HH, May PC, Finch CE. Effective oral administration of 17 beta-estradiol to female C57BL/6J mice through the drinking water. Biol Reprod. 1986;35:1088–95. PubMed

Haslam SZ, Shyamala G. Effect of oestradiol on progesterone receptors in normal mammary glands and its relationship with lactation. Biochem J. 1979;182:127–31. PubMed PMC

Barnett KR, Schilling C, Greenfeld CR, Tomic D, Flaws JA. Ovarian follicle development and transgenic mouse models. Hum Reprod Update. 2006;12:537–55. PubMed

MacLennan MB, Anderson BM, Ma DWL. Differential mammary gland development in FVB and C57Bl/6 mice: implications for breast cancer research. Nutrients. 2011;3:929–36. PubMed PMC

Aupperlee MD, Drolet AA, Durairaj S, Wang W, Schwartz RC, Haslam SZ. Strain-specific differences in the mechanisms of progesterone regulation of murine mammary gland development. Endocrinology. 2009;150:1485–94. PubMed PMC

Chia R, Achilli F, Festing MFW, Fisher EMC. The origins and uses of mouse outbred stocks. Nat Genet. 2005;37:1181–6. PubMed

Kocieniewski P, Lipniacki T. MEK1 and MEK2 differentially control the duration and amplitude of the ERK cascade response. Phys Biol. 2013;10:035006. PubMed

Ryu H, Chung M, Dobrzyński M, Fey D, Blum Y, Lee SS, et al. Frequency modulation of ERK activation dynamics rewires cell fate. Mol Syst Biol. 2015;11:838. PubMed PMC

Curtis Hewitt S, Couse JF, Korach KS. Estrogen receptor transcription and transactivation: estrogen receptor knockout mice: what their phenotypes reveal about mechanisms of estrogen action. Breast Cancer Res. 2000;2:345–52. PubMed PMC

Conneely OM, Mulac-Jericevic B, Lydon JP, De Mayo FJ. Reproductive functions of the progesterone receptor isoforms: lessons from knock-out mice. Mol Cell Endocrinol. 2001;179:97–103. PubMed

Mulac-Jericevic B, Lydon JP, DeMayo FJ, Conneely OM. Defective mammary gland morphogenesis in mice lacking the progesterone receptor B isoform. Proc Natl Acad Sci U S A. 2003;100:9744–9. PubMed PMC

Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery CA, et al. Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev. 1995;9:2266–78. PubMed

Jerry DJ, Shull JD, Hadsell DL, Rijnkels M, Dunphy KA, Schneider SS, et al. Genetic variation in sensitivity to estrogens and breast cancer risk. Mamm Genome. 2018;29:24–37. PubMed PMC

Wadia PR, Vandenberg LN, Schaeberle CM, Rubin BS, Sonnenschein C, Soto AM. Perinatal bisphenol A exposure increases estrogen sensitivity of the mammary gland in diverse mouse strains. Environ Health Perspect. 2007;115:592–8. PubMed PMC

Ferraj A, Audano PA, Balachandran P, Czechanski A, Flores JI, Radecki AA, et al. Resolution of structural variation in diverse mouse genomes reveals chromatin remodeling due to transposable elements. Cell Genomics. 2023;3:100291. PubMed PMC

Lilue J, Doran AG, Fiddes IT, Abrudan M, Armstrong J, Bennett R et al. Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci. Nat Genet. 2018;50(11):1574–83. PubMed PMC

CD-1®. IGS Mouse | Charles River.

Lavoie H, Gagnon J, Therrien M. ERK signalling: a master regulator of cell behaviour, life and fate. Nat Rev Mol Cell Biol. 2020;21(10):607–32. PubMed

Sternlicht MD, Sunnarborg SW, Kouros-Mehr H, Yu Y, Lee DC, Werb Z. Mammary ductal morphogenesis requires paracrine activation of stromal EGFR via ADAM17-dependent shedding of epithelial amphiregulin. Development. 2005;132:3923–33. PubMed PMC

Mallepell S, Krust A, Chambon P, Brisken C. Paracrine signaling through the epithelial estrogen receptor alpha is required for proliferation and morphogenesis in the mammary gland. Proc Natl Acad Sci U S A. 2006;103:2196–201. PubMed PMC

Aoki K, Kumagai Y, Sakurai A, Komatsu N, Fujita Y, Shionyu C, et al. Stochastic ERK activation induced by noise and cell-to-cell propagation regulates cell density-dependent proliferation. Mol Cell. 2013;52:529–40. PubMed

Crozet F, Levayer R. Emerging roles and mechanisms of ERK pathway mechanosensing. Cell Mol Life Sci. 2023;80:355. PubMed PMC

Koledova Z, Lu P. A 3D fibroblast-epithelium co-culture model for understanding Microenvironmental Role in branching morphogenesis of the mammary gland. Methods Mol Biol. 2017;1501:217–31. PubMed

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82. PubMed PMC

Bankhead P, Loughrey MB, Fernández JA, Dombrowski Y, McArt DG, Dunne PD, et al. QuPath: open source software for digital pathology image analysis. Sci Rep. 2017;7:16878. PubMed PMC

Methods and Models in Mammary Gland Biology and Breast Cancer Research