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Reporter system development form genome instability in C. elegans

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Reporter system development form genome instability in C. elegans

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Genome instability can lead to changes in the genome and thereby disturb normal cell functions. This can result in cancer. To study genome instability the nematode C. elegans can be used. This report describes the development of reporter systems for 3 forms of genome instability in C. elegans. These reporter systems can be used to identify genes that are involved in the maintenance of genome integrity.
Base-base mispairing
During replication base-base mispairs can occur, when this is not repaired it can lead to a permanent mutation. The mismatch repair system is able to recognize these mispairs and replace them with the correct base. To develop a reporter system for base-base mispairing, LacZ gene was used as a reporter gen. In this gene a stop codon was added. Only when this stop codon is converted by a mutation, transcription can proceed. Transgenic lines were made by micro-injection in C. elegans and functionality of the reporter was tested by knocking down the expression of mismatch repair gene msh-2 by RNA interference. These results showed that the constructed plasmid was able to express functional LacZ, even when the stop codon was not converted by mutations. This requires further experimentation.
Microsatellite instability
Another form of genome instability is microsatellite instability. Mono-, di-, tri-, or tetranucleotide repeats are sensitive to replication slippage and can cause frame-shifts. A reporter system for microsatellite instability was improved by replacing the original heat shock promoter for a myo-2 promoter. This reporter plasmid has an ‘out frame’ LacZ gene containing a C23 stretch. LacZ can only be expressed when a frame-shift occurs and the LacZ gene shifts to an ‘in frame’ orientation. The functionality of this new reporter system was comparable with the original system when the mutation frequency was elevated by RNA interference of the gene msh-2.
G-tract instability
It has been hypothesized that when poly-guanine tracts are present in the lagging strand during replication, secondary structures that block replication can be formed. This can result in deletions upstream of the G-tract. The reporter plasmid for microsatellite instability is converted to a G-tract instability reporter plasmid by creating identical sequences upstream and downstream of the C23 stretch and incorporation of a stop codon downstream of the stretch. The identical sequences are presumed to elevate levels of LacZ restoration. Transgenic lines were made with this reporter plasmid, but could not be tested because of time limitations.

Toon meer
OrganisatieHogeschool Utrecht
OpleidingBiologie en Medisch Laboratorium Onderzoek
AfdelingLife Sciences en Chemistry
PartnersHubrecht Institute
Jaar2008
TypeBachelor
TaalEngels

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