dc.contributor.advisor | Lobert, Viola | |
dc.contributor.author | Sagerud, Michael Venbakken | |
dc.contributor.author | Strand, Sebastian Rishaug | |
dc.date.accessioned | 2022-06-21T06:55:20Z | |
dc.date.available | 2022-06-21T06:55:20Z | |
dc.date.issued | 2022 | |
dc.identifier.uri | https://hdl.handle.net/11250/2999706 | |
dc.description.abstract | Type 1-diabetes is a disease where the body destroys its own insulin-producing, pancreatic beta cells. The pancreatic
alpha cells produce the blood sugar-elevating hormone glucagon from the precursor protein proglucagon.
Marking the proglucagon gene with CRISPR/Cas9 mediated knock-in of green fluorescent protein (GFP) might allow for
visualising the dynamics within the cell under differentiation from stem cell to somatic cell.
To perform the preferred knock-in, the gene editing tools were constructed. A combined vector of single guide RNA,
Cas9 nuclease and a reporter for the Cas9 nuclease was constructed. A donor DNA template vector consisting of the
GFP between sequences homologous to each side of the Cas9 cut site was cloned with In-Fusion multi-fragment
cloning. Sanger sequencing confirmed that both vectors were successfully cloned before the combined vector was
successfully transfected into human embryonic kidney cells. T7 Endonuclease I assay estimated an indel occurrence of
14,4% for the combined vector.
The combined vector showed that it did perform cutting, and future research will reveal if the constructed donor DNA
template vector results in a knock-in of GFP in the proglucagon gene. | en_US |
dc.language.iso | eng | en_US |
dc.publisher | OsloMet-Storbyuniversitetet | en_US |
dc.subject | CRISPR/Cas9 | en_US |
dc.subject | Type 1-diabetes | en_US |
dc.subject | Green fluorescent protein reporter gene | en_US |
dc.title | CRISPR/Cas9 gene-editing tools construction | en_US |
dc.type | Bachelor thesis | en_US |
dc.description.version | publishedVersion | en_US |