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Simultaneous detection of Homologous Recombination and Non-Homologous End Joining in eukaryotic cells

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Simultaneous detection of Homologous Recombination and Non-Homologous End Joining in eukaryotic cells

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All cells contain proteins, which perform all functions inside the cell. Proteins are coded in DNA and are created by transcription and translation of the DNA. This only functions properly when the DNA is intact. Damage in the DNA can cause missing or mutated genes, which code for faulty proteins, which in turn can lead to apoptosis or carcinogenesis. Double strand breaks (DSB) are one of the most dangerous forms of DNA damage. Double strand breaks can lead to chromosome loss and/or chromosome rearrangements, in turn leading to gene loss or mutations.
The project was focused on two major DSB repair pathways: Homologous Recombination and Non-Homologous End Joining. There are several methods to detect and/or measure each pathway, but there is not yet an assay to detect both pathways at once. This is required to study the balance between the two pathways and ultimately to test the effect of knock-out genes to study their influence on both pathways.
For this purpose a reporter construct has been designed. This construct utilizes Sleeping Beauty (SB) Transposase to introduce a single DSB which the cell needs to repair. Repair results in the expression of one or two fluorescent proteins, with different localizations, dependent on the repair pathway used.
Currently, the construct has been stably integrated into cells, however, no repair events have been observed.
Several experiments have been performed to find the cause for the lack of repair. One possibility was transposase inactivity. To test this the transposase-expression plasmid, more specifically the transposase open reading frame (ORF), has been sequenced and this resulted in the discovery of three missing nucleotides when compared to other SB transposase-expression plasmids. The ORF has been repaired and the plasmid has been transfected, but still no transposase activity has been detected.

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OrganisatieAvans Hogeschool
OpleidingBiologie en Medisch Laboratoriumonderzoek-Breda
AfdelingATGM Academie voor de technologie van Gezondheid en Milieu
PartnersErasmus Medisch Centrum
Datum2014-01-06
TypeBachelor
TaalEngels

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