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Danielsson, Louise, 2007. RNA interference : optimizing a protocol for HC11 cells. SLU, Dept. of Biomedical Sciences and Veterinary Public Health, Uppsala. Uppsala: SLU, Dept. of Biomedical Sciences and Veterinary Public Health

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Abstract

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.

Main title:RNA interference
Subtitle:optimizing a protocol for HC11 cells
Authors:Danielsson, Louise
Supervisor:Tallkvist, Jonas
Examiner:UNSPECIFIED
Series:UNSPECIFIED
Volume/Sequential designation:UNSPECIFIED
Year of Publication:2007
Level and depth descriptor:Other
Student's programme affiliation:BTEMP Biotechnology Programme 240 HEC
Department:(VH) > Dept. of Biomedical Sciences and Veterinary Public Health
Keywords:RNA Interference, HC11 cells
URN:NBN:urn:nbn:se:slu:epsilon-s-7633
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-s-7633
Subjects:SLU > (VH) > Dept. of Biomedical Sciences and Veterinary Public Health
Veterinary science and hygiene - General aspects
Language:English
Deposited On:09 Oct 2017 09:50
Metadata Last Modified:09 Oct 2017 09:50

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