The PinPoint Targeted Integration system allows the easy and efficient production of isogenic stable cell lines in mammalian and other cell types. Custom gene expression cassettes can be engineered into target genomes using the unique PinPoint integrase with site-specific control. This technology enables the generation of platform cell lines which can be used to routinely knock-in different transgenes and reporters at the same genetic locus in cells with the same genetic background. This level of targeting control allows for the study of phenotypic effects free from context and positional variations, which results in more accurate genotype to phenotype correlations.
The PinPoint system is a two-step approach for engineering of target cells with an optional third step for selection cassette removal by Cre resolvase.
The first step involves insertion of a plasmid bearing the PinPoint placement site via transfection into the target cell genome. This can be done using the PinPoint-FC system that involves the well-characterized phiC31 integrase system.
The second part of the PinPoint system relies on the introduction of a donor vector containing your desired gene cassette insert, which is integrated into the placed PinPoint site using a hyperspecific and efficient PinPoint integrase. The PinPoint integrase catalyzes the attB x attP reaction between the placed site (attP) and the attB site in the donor vector to insert the donor vector at the placed site each and every targeting event.
The third optional step invloves the removal of the entire backbone (excluding the insert and its promoter) using the well-characterized Cre/LoxP reaction leaving only the promoter/insert combination (and a single LoxP site) in the genome.
Platform cell lines containing the PinPoint attP site can be made through two different approaches. The PinPoint-FC system utilizes the phiC31 integrase along with the PinPoint-FC attP Placement Vector. This will integrate the cassette at pseudo attP sites within the targeted genome with typically single copy insertions.
Once the PinPoint attP site has been successfully placed into the desired genomic site, this site can then be efficiently and routinely targeted for site-specific integration of expression cassettes at that locus. The expression cassette can be driven by EF1a (PIN500A-1-SBI), CAGs (PIN510A-1-SBI), or a custom-designed promoter of your choice (PIN520A-1-SBI). Once the gene or reporter of interest has been cloned into the PinPoint Integrase Donor Vector, a simple co-transfection of the placed attP site cell line with the PinPoint Integrase Vector will result in a specific recombination reaction between the PinPoint attP site and the attB site in the donor vector construct.
PinPoint integrase and donor vectors
PinPoint-FC 293T Platform Kit for Targeted Gene Insertion (PinPoint attP already placed) (1 step)
PinPoint-FC Murine iPSC Platform Kit for Targeted Gene Insertion (PinPoint attP already placed) (1 step)