Optimalized transient transfection of chondrogenic primary cell cultures |
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Authors: | Tamás Juhász Csaba Matta Zoltán Mészár Georgina Nagy Zsolt Szíjgyártó Zsanett Molnár Bernadett Kolozsvári Éva Bakó Róza Zákány |
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Institution: | (1) Red Cross Blood Transfusion Service of Upper Austria, Blumauerstr. 3-5, Linz, A-4020, Austria;(2) Ludwig Boltzmann Institute for Experimental and Clinical Traumatology/AUVA Research Centre, Linz/Vienna, Austria;(3) Bio-Products & Bio-Engineering AG, Vienna, Austria;(4) Institute for Applied Microbiology, University of Natural Resources and Applied Life Sciences, Vienna, Austria;(5) Department of Human Genetics – General Women’s and Children’s Hospital, Linz, Austria |
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Abstract: | We aimed to find a transfection method which provides high efficiency with minimal cytotoxic and/or apoptotic effects for
gene transfer into multilayer primary chondrogenic cell cultures. The pEGFP-C1 plasmid was introduced into the cell culture
and the efficiency of transformation quantified by GFP fluorescence; the resulting nucleofection was effective but resulted
in severe apoptosis. Two liposomal reagents designed to allow transfection into adherent cells did not deliver the plasmids
sufficiently and cartilage formation did not occur. In addition, a third liposomal compound, recommended for transfection
into either adherent or suspension cell cultures, lead to acceptable transfection efficiency but no cartilage formation. When
an amphiphilic reagent was used however, there was acceptable transfection efficiency as well as cartilage formation. The
viability of the cells which were transfected using the amphiphilic reagent remained unaffected but proliferation was severely
diminished, particularly in the presence of GFP. In addition, the amount of cartilage decreased when GFP was expressed, despite
unchanged levels of mRNAs of sox9 and aggrecan core protein, factors reflecting on the efficiency of chondrogenesis. Overexpression of both the constitutively
active delta and gamma isoforms of catalytic subunit of calcineurin, a protein phosphatase described as a positive regulator
of chondrogenesis, decreased protein level of Sox9 and subsequent cartilage formation. In conclusion, we found that amphiphilic
reagent applied prior to the adhesion of cells provides a useful means to transfer plasmids to primary differentiating chondrogenic
cells. |
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