| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | The Cas9-GFP-geminin plasmid was generated by subcloning to produce a recombinant Cas9-GFP-geminin fusion protein. The backbone blast is Lv5-EF1α-Cas9-Blast (MilliporeSigma, Lot# LVCAS9BST), which contains the unique Xbal (NEB, R0145T) and HpaI (NEB, R0105S) restriction enzyme sites used in cloning. The geminin gene inserted was carried by a pUC57 plasmid vector from GenScript (product# 648334-1). This gene was cut from the parent plasmid using Xbal (NEB, R0145T) and HpaI (NEB, R0105S) restriction enzymes. The backbone was cut using the same enzymes, creating compatible ends. The geminin insert was then ligated into the backbone to create a Cas9-GFP-geminin fusion protein expression vector. The Cas9 only plasmid used was a product of Cas9GFP (MilliporeSigma, Lot# 05271523MN), which uses a CMV promoter for strong, transient expression of Cas9. | Get A Quote |
Targeting specific chromosomal sequences for genome modification or regulation during particular phases of the cell cycle may prove useful in creating more precise, predictable genetic changes. Here, we present a system using a fusion protein comprised of a programmable DNA modification protein, Cas9, linked to a cell cycle regulated protein, geminin, as well as green fluorescent protein (GFP) for visualization. Despite the large size of Cas9 relative to geminin, cells were observed to express Cas9-GFP-geminin at levels which oscillate with the cell cycle. These fusion proteins are also shown to retain double-strand break (DSB) activity at specific chromosomal sequences to produce both indels and targeted integ... More
