{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T23:23:39Z","timestamp":1792020219194,"version":"4.4.0"},"reference-count":43,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2020,1,8]],"date-time":"2020-01-08T00:00:00Z","timestamp":1578441600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Startup Program from Foshan University","award":["gg040968"],"award-info":[{"award-number":["gg040968"]}]},{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","doi-asserted-by":"publisher","award":["400259"],"award-info":[{"award-number":["400259"]}],"id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Granulosa cells (GCs) are the key components of ovarian follicles for regulating oocyte maturation. Previous established GC lines have allowed prolonged proliferation, but lost some physiological features owing to long-term immortalization. This study was to establish an induced immortal porcine GC line with reversible proliferation status by the tetracycline inducible (Tet-on) 3G system. Our conditional immortal porcine GCs (CIPGCs) line steadily propagated for at least six months and displayed primary GC morphology when cultured in the presence of 50 ng\/mL doxycycline [Dox (+)]. Upon Dox withdrawal [Dox (\u2013)], Large T-antigen expression, reflected by mCherry fluorescence, gradually became undetectable within 48 h, accompanied by less proliferation and size increase. The levels of estradiol and progesterone, and the expression of genes associated with steroid production, such as CYP11A1 (cytochrome P450 family 11), 3\u03b2-HSD (3\u03b2-hydroxysteroid dehydrogenase), StAR (steroidogenic acute regulatory protein), and CYP19A1 (cytochrome P450 family 19 subfamily a member 1), were all significantly higher in the Dox (\u2013) group than Dox (+) group. The CIPGCs could switch into a proliferative state upon Dox induction. Interestingly, the expression of StAR and CYP19A1 in the CIPGCs (\u2013Dox) was significantly increased by adding porcine follicular fluid (PFF) to mimic an ovary follicle environment. Moreover, PFF priming the CIPGCs in Dox (\u2013) group resulted in similar estradiol production as that of primary GC, and enabled this cell line to respond to gonadotrophins in estradiol production. Collectively, we have established an inducible immortal porcine GC line, which offers a unique and valuable model for future research on the regulation of ovarian functions.<\/jats:p>","DOI":"10.3390\/cells9010156","type":"journal-article","created":{"date-parts":[[2020,1,9]],"date-time":"2020-01-09T03:07:11Z","timestamp":1578539231000},"page":"156","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Establishment of A Reversibly Inducible Porcine Granulosa Cell Line"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6482-0281","authenticated-orcid":false,"given":"Yinshan","family":"Bai","sequence":"first","affiliation":[{"name":"School of Life Science and Engineering, Foshan University, Foshan 528231, China"},{"name":"Department of Animal Biosciences, University of Guelph, Guelph, ON N1G 2W1, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cui","family":"Zhu","sequence":"additional","affiliation":[{"name":"School of Life Science and Engineering, Foshan University, Foshan 528231, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meiying","family":"Feng","sequence":"additional","affiliation":[{"name":"College of Animal Science, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0984-2058","authenticated-orcid":false,"given":"Bo","family":"Pan","sequence":"additional","affiliation":[{"name":"Department of Animal Biosciences, University of Guelph, Guelph, ON N1G 2W1, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shouquan","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Animal Science, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8479-9320","authenticated-orcid":false,"given":"Xiaoshu","family":"Zhan","sequence":"additional","affiliation":[{"name":"Department of Animal Biosciences, University of Guelph, Guelph, ON N1G 2W1, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huifang","family":"Chen","sequence":"additional","affiliation":[{"name":"School of Life Science and Engineering, Foshan University, Foshan 528231, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bingyun","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Life Science and Engineering, Foshan University, Foshan 528231, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julang","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Animal Biosciences, University of Guelph, Guelph, ON N1G 2W1, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,1,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"649","DOI":"10.1007\/s00441-012-1386-8","article-title":"Follicular determinants of pregnancy establishment and maintenance","volume":"349","author":"Pohler","year":"2012","journal-title":"Cell Tissue Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/j.jsbmb.2014.02.013","article-title":"Knockdown of CEBPbeta by RNAi in porcine granulosa cells resulted in S phase cell cycle arrest and decreased progesterone and estradiol synthesis","volume":"143","author":"Zhen","year":"2014","journal-title":"J. Steroid Biochem. Mol. Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1093\/molehr\/2.6.391","article-title":"Expression of Ad4-BP\/cytochrome P450 side chain cleavage enzyme and induction of cell death in long-term cultures of human granulosa cells","volume":"2","author":"Breckwoldt","year":"1996","journal-title":"Mol. Hum. Reprod."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"29","DOI":"10.2302\/kjm.54.29","article-title":"Establishment of an immortalized porcine granulosa cell line (PGV) and the study on the potential mechanisms of PGV cell proliferation","volume":"54","author":"Lin","year":"2005","journal-title":"Keio J. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/0303-7207(93)90059-S","article-title":"Characterization of progesterone biosynthesis in a transformed granulosa cell line","volume":"94","author":"Rao","year":"1993","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1262\/jrd.2016-111","article-title":"An immortalized steroidogenic goat granulosa cell line as a model system to study the effect of the endoplasmic reticulum (ER)-stress response on steroidogenesis","volume":"63","author":"Yang","year":"2017","journal-title":"J. Reprod. Dev."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"R31","DOI":"10.1530\/REP-15-0546","article-title":"Establishment and applications of male germ cell and sertoli cell lines","volume":"152","author":"Wang","year":"2016","journal-title":"Reproduction"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1677\/joe.1.05718","article-title":"A steroidogenic cell line with differentiation potential from mouse granulosa cells, transfected with Ad4BP and SV40 large T antigen genes","volume":"185","author":"Kamei","year":"2005","journal-title":"J. Endocrinol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1002\/1098-2795(200012)57:4<366::AID-MRD9>3.0.CO;2-B","article-title":"Generation of stable cell lines by spontaneous immortalization of primary cultures of porcine granulosa cells","volume":"57","author":"Swan","year":"2000","journal-title":"Mol. Reprod. Dev."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1016\/j.mce.2004.04.018","article-title":"Ovarian granulosa cell lines","volume":"228","author":"Havelock","year":"2004","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_11","first-page":"59","article-title":"SV40 T antigen and telomerase are required to obtain immortalized human adult bone cells without loss of the differentiated phenotype","volume":"13","author":"Darimont","year":"2002","journal-title":"Cell Growth. Differ."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"184","DOI":"10.14712\/fb2015061050184","article-title":"Characterization of porcine granulosa cell Line AVG-16","volume":"61","author":"Sadowska","year":"2015","journal-title":"Folia Biol. (Praha)"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/0303-7207(93)90208-2","article-title":"Expression of the IGF system in primary and immortalized porcine ovarian granulosa cells","volume":"97","author":"Leighton","year":"1993","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1002\/(SICI)1098-2795(199611)45:3<299::AID-MRD6>3.0.CO;2-N","article-title":"Steroidogenic properties of a spontaneously established porcine granulosa cell line (PGC-2)","volume":"45","author":"Kwan","year":"1996","journal-title":"Mol. Reprod. Dev."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1016\/S0303-7207(98)00233-0","article-title":"Regulation of steroidogenesis in jc-410, a stable cell line of porcine granulosa origin","volume":"148","author":"Rodway","year":"1999","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"5950","DOI":"10.1038\/sj.onc.1207801","article-title":"Cre-mediated reversible immortalization of human renal proximal tubular epithelial cells","volume":"23","author":"Kowolik","year":"2004","journal-title":"Oncogene"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1016\/j.jbiotec.2010.12.003","article-title":"Reversible immortalization of rat pancreatic beta cells with a novel immortalizing and tamoxifen-mediated self-recombination tricistronic vector","volume":"151","author":"Wu","year":"2011","journal-title":"J. Biotechnol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/j.scr.2018.01.017","article-title":"Establishment and characterization of a human embryonic stem cell line, NERCe002-A-3, with inducible 14-3-3zeta overexpression","volume":"28","author":"He","year":"2018","journal-title":"Stem. Cell Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1016\/j.jbiotec.2011.05.010","article-title":"Establishment of an Atlantic salmon kidney cell line with an inducible gene expression system","volume":"154","author":"Collet","year":"2011","journal-title":"J. Biotechnol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3589","DOI":"10.1172\/JCI58447","article-title":"A genetically engineered human pancreatic beta cell line exhibiting glucose-inducible insulin secretion","volume":"121","author":"Ravassard","year":"2011","journal-title":"J. Clin. Investig."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"916","DOI":"10.1016\/j.molmet.2015.09.008","article-title":"A human beta cell line with drug inducible excision of immortalizing transgenes","volume":"4","author":"Benazra","year":"2015","journal-title":"Mol. Metab."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1171","DOI":"10.1016\/j.csbj.2019.07.015","article-title":"Drug inducible CRISPR\/Cas systems","volume":"17","author":"Zhang","year":"2019","journal-title":"Comput. Struct. Biotechnol. J."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"423","DOI":"10.3892\/ijmm.2016.2658","article-title":"Differential gene expression in mouse spermatogonial stem cells and embryonic stem cells","volume":"38","author":"Bai","year":"2016","journal-title":"Int. J. Mol. Med."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"10166","DOI":"10.1074\/jbc.M111.293274","article-title":"Follicle-stimulating hormone regulates pro-apoptotic protein Bcl-2-interacting mediator of cell death-extra long (BimEL)-induced porcine granulosa cell apoptosis","volume":"287","author":"Wang","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_25","first-page":"1575","article-title":"Prognostic value of serum and ascites levels of estradiol, FSH, LH and prolactin in ovarian cancer","volume":"29","author":"Chen","year":"2009","journal-title":"Anticancer Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1159\/000453394","article-title":"Establishment and characterization of an SV40 Large T antigen-transduced porcine colonic epithelial cell line","volume":"203","author":"Kaiser","year":"2017","journal-title":"Cells Tissues Organs"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Chen, Y., Hu, S., Wang, M., Zhao, B., Yang, N., Li, J., Chen, Q., Liu, M., Zhou, J., and Bao, G. (2019). Characterization and Establishment of an immortalized rabbit melanocyte cell Line using the SV40 Large T antigen. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20194874"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"16922","DOI":"10.1038\/srep16922","article-title":"Establishment and Characterization of human germline stem cell line with unlimited proliferation potentials and no tumor formation","volume":"5","author":"Hou","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"714","DOI":"10.1007\/s11626-015-9885-z","article-title":"Establishment of immortalized murine mesothelial cells and a novel mesothelioma cell line","volume":"51","author":"Blum","year":"2015","journal-title":"In Vitr. Cell. Dev. Biol. Anim."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"800","DOI":"10.1042\/bst0300800","article-title":"Conditionally immortalized cell lines as model systems for high-throughput biology in drug discovery","volume":"30","author":"Daniele","year":"2002","journal-title":"Biochem. Soc. Trans."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.exer.2018.07.011","article-title":"Establishment of a conditionally immortalized mouse optic nerve astrocyte line","volume":"176","author":"Liu","year":"2018","journal-title":"Exp. Eye Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1080\/21623945.2014.995510","article-title":"Conditional immortalization of primary adipocyte precursor cells","volume":"4","author":"Church","year":"2015","journal-title":"Adipocyte"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1074","DOI":"10.1002\/eji.201747358","article-title":"Fast and efficient genetic engineering of hematopoietic precursor cells for the study of dendritic cell migration","volume":"48","author":"Leithner","year":"2018","journal-title":"Eur. J. Immunol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"5637","DOI":"10.1210\/en.2012-1556","article-title":"Transient, inducible, placenta-specific gene expression in mice","volume":"153","author":"Fan","year":"2012","journal-title":"Endocrinology"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/S1084-9521(02)00018-6","article-title":"Tetracycline-controlled transcriptional regulation systems: Advances and application in transgenic animal modeling","volume":"13","author":"Zhu","year":"2002","journal-title":"Semin. Cell Dev. Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2885","DOI":"10.3748\/wjg.v11.i19.2885","article-title":"Generation of the regulatory protein rtTA transgenic mice","volume":"11","author":"Xu","year":"2005","journal-title":"World J. Gastroenterol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1262\/jrd.2017-048","article-title":"Age-associated changes in granulosa cells and follicular fluid in cows","volume":"63","author":"Iwata","year":"2017","journal-title":"J. Reprod. Dev."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1002\/biot.201500236","article-title":"Selecting the optimal Tet-On system for doxycycline-inducible gene expression in transiently transfected and stably transduced mammalian cells","volume":"11","author":"Das","year":"2016","journal-title":"Biotechnol. J."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1093\/hmg\/ddg017","article-title":"Human BOULE gene rescues meiotic defects in infertile flies","volume":"12","author":"Xu","year":"2003","journal-title":"Hum. Mol. Genet."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"7414","DOI":"10.1073\/pnas.131090498","article-title":"A gene family required for human germ cell development evolved from an ancient meiotic gene conserved in metazoans","volume":"98","author":"Xu","year":"2001","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1382","DOI":"10.1038\/sj.gt.3302780","article-title":"Optimization of the Tet-On system for regulated gene expression through viral evolution","volume":"13","author":"Zhou","year":"2006","journal-title":"Gene. Ther."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1016\/S0960-0760(99)00028-X","article-title":"Steroid regulation of progesterone synthesis in a stable porcine granulosa cell line: A role for progestins","volume":"68","author":"Rodway","year":"1999","journal-title":"J. Steroid. Biochem. Mol. Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"712","DOI":"10.1016\/j.chembiol.2017.05.012","article-title":"Biochemical mechanisms and catabolic enzymes involved in bacterial estrogen degradation pathways","volume":"24","author":"Chen","year":"2017","journal-title":"Cell Chem. Biol."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/9\/1\/156\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T13:42:30Z","timestamp":1760362950000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/9\/1\/156"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,1,8]]},"references-count":43,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2020,1]]}},"alternative-id":["cells9010156"],"URL":"https:\/\/doi.org\/10.3390\/cells9010156","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,1,8]]}}}