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Optimization and evaluation of electroporation delivery of siRNA in the human leukemic CEM cell line
Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
2010 (English)In: CYTOTECHNOLOGY, ISSN 0920-9069, Vol. 62, no 6, 497-507 p.Article in journal (Refereed) Published
Abstract [en]

In order to study nucleoside analog activation in the CEM cell line, a transfection protocol had to be optimized in order to silence an enzyme involved in nucleoside analog activation. Hematopoetic cell lines can be difficult to transfect with traditional lipid-based transfection, so the electroporation technique was used. Field strength, pulse length, temperature, electroporation media, siRNA concentration, among other conditions were tested in order to obtain approximately 70-80% mRNA and enzyme activity downregulation of the cytosolic enzyme deoxycytidine kinase (dCK), necessary for nucleoside analog activation. Downregulation was assessed at mRNA and enzyme activity levels. After optimizing the protocol, a microarray analysis was performed in order to investigate whether the downregulation was specific. Additionally two genes were differentially expressed besides the downregulation of dCK. These were however of unknown function. The leakage of intracellular nucleotides was also addressed in the electroporated cells since it can affect the DNA repair mechansism and the efficiency of nucleoside analogs. Three of these pools were increased compared to untreated, unelectroporated cells. The siRNA transfected cells with reduced dCK expression and activity showed reduced sensitivity to several nucleoside analogs as expected. The multidrug resistance to other drugs, as seen in nucleoside analog-induced resistant cells, was not seen with this model.

Place, publisher, year, edition, pages
Springer Science Business Media , 2010. Vol. 62, no 6, 497-507 p.
Keyword [en]
RNAi, Electroporation, Microarray, Deoxycytidine kinase, Nucleoside analog
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-63397DOI: 10.1007/s10616-010-9309-6ISI: 000284844300003OAI: oai:DiVA.org:liu-63397DiVA: diva2:379143
Note
The original publication is available at www.springerlink.com: Anna Fyrberg and Kourosh Lotfi, Optimization and evaluation of electroporation delivery of siRNA in the human leukemic CEM cell line, 2010, CYTOTECHNOLOGY, (62), 6, 497-507. http://dx.doi.org/10.1007/s10616-010-9309-6 Copyright: Springer Science Business Media http://www.springerlink.com/ Available from: 2010-12-17 Created: 2010-12-17 Last updated: 2011-01-13

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Fyrberg, AnnaLotfi, Kourosh

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