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Danielsson, Louise (2007) RNA interference. Other thesis, SLU.

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RNA interference is a naturally occurring process of gene silencing that reduces the expression of the gene from which the RNA sequence is derived, when added in a double-stranded form. RNA interference works as defense against viruses and transposons. The aim of this study was to optimize a protocol for RNA interference in mouse mammary epithelial HC11 cells by using different controls, and finally to silence the drug transporter BCRP to do an uptake-study with the BCRP-substrate mitoxantrone. The optimization of the protocol was performed with four different lipids, as well as sicontrol tox, negative control and a positive control Cyclophilin B. Sicontrol tox and the negative control were assessed with ATP determination. Real time PCR was performed to check the down-regulation of Cyclophilin B after RNAi treatment of the HC11 cells. The results of the optimization protocol showed that transfection for 48 h in the presence of lipid nr 1 diluted 1:4 times works best for transfection in HC11 cells. Cyclophilin B was down-regulated about 90 % with this protocol, and BCRP was down-regulated 80 %. The uptake-study with mitoxantrone showed that the BCRP gene silenced cells accumulated the double amount of mitoxantrone as compared to controls. In conclusion, the obtained results provide a promising model for screening BCRP substrates, which can be used to examine other transporters in mammary epithelial cells, as well.

Item Type: Thesis (Other)
Keywords: RNA Interference, HC11 cells
Subject (faculty): Faculty of Veterinary Medicine and Animal Science > Department of Biomedical Science and Veterinary Public Health
Divisions: SLU > Faculty of Veterinary Medicine and Animal Science
Depositing User: Louise Danielsson
Date Deposited: 18 Jun 2008
Last Modified: 18 Aug 2015 09:52
URI: http://ex-epsilon.slu.se/id/eprint/1811

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