A versatile chondrogenic rat calvaria cell line R-tTA-24 that permits tetracycline-regulated gene expression |
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| Authors: | C Bergwitz T Wendlandt E Pötter I Glomb K Gras A von zur Mühlen G Brabant |
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| Institution: | (1) Abt. für Klinische Endokrinologie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany, DE;(2) Heritable Disorders Branch, NICHD/NIH, Center Drive, Bethesda, MD 20892, USA e-mail: bergwitc@mail.nih.gov, US |
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| Abstract: | The clonal rat calvaria cell line RCJ3.1C5.18 (RCJ) undergoes chondrogenic differentiation after long-term culture post confluence.
To allow flexible genetic manipulation, a tetracycline-regulated gene expression system was established in this cell line.
Treatment with tetracycline in operational doses does not affect the differentiation of RCJ cells with respect to the markers
tested. After stable transfection with pUHD15.1 containing the tetracycline transactivator (tTA) in the presence of pTK-hyg
for hygromycin selection, 28 clones were isolated and characterized for alcian blue staining of cartilage-specific proteoglycans
and for collagen type II expression. Clone R-tTA-24 was selected on the basis of phenotype and displayed tetracycline-dependent
downregulation of luciferase activity (tet-OFF system) by two orders of magnitude (57–149-fold) after stable transfection
with the reporter gene pBI-EGFP/luc. The novel, chondrogenic cell line R-tTA-24 may be stably transfected with various genes
of interest for tetracycline- regulated gene expression using neomycin selection and may be a valuable tool to study the process
of chondrogenic differentiation in vitro.
Accepted: 30 November 1999 |
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