Líffræðifélag Íslands - biologia.is
Líffræðiráðstefnan 2021
Erindi/veggspjald / Talk/poster V45
Höfundar / Authors: Birkir Orri Viðarsson, Jón Baldvin Helgason Möller, Stefán Eggertsson, Zophonías O. Jónsson and Sigríður Rut Franzdóttir
Starfsvettvangur / Affiliations: Líf- og umhverfisvísindastofnun Háskóla Íslands
Kynnir / Presenter: Jón Baldvin Helgason Möller og Birkir Orri Viðarsson
Live imaging of cellular processes at high resolution requires a palette of fluorescently tagged proteins. In multicellular organisms, ectopic expression of genes can result in unwanted phenotypes and the same is often true in transfected cells. CRISPR mediated genome editing provides an option to insert knock-in tags on genes at the relevant genomic locus, and although this may in some cases result in abnormal gene function, provides far more appropriate measure of native gene behavior than over-expression. For knock-in by homology directed recombination, donor constructs containing the tag of interest flanked by homology arms (ranging from ~60-1000 bp in length, depending on the experimental system) must be generated. While synthetic DNA can be generated for shorter homology arms, longer arms call for molecular assembly cloning of up to 4 fragments (5, if a long linker is added). This process repeated for multiple genes and fluorescent tags is expensive and time-consuming. Therefore, we set out to simplify the process by generating a set of vectors for picking up the fluorescent protein and linker from one plasmid, and dropping into pre-donor constructs containing continuous homology with the desired target region. We call this system pPickUp. The PickUp plasmids are universal, and apart from potential codon usage issues, can be used to quickly generate a color-palette of donor constructs for C-terminal fusions of any gene of interest from any species.