Efficiently create isogenic stable cell lines
- Create stable isogenic cell lines at defined loci
- High efficiency insertion with no size limit
- Avoid gene integration site variation
- High-throughput cell line engineering
The PinPoint Targeted Integration System
The PinPoint Targeted Integration system allows users to easily and efficiently create 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 integrase can be used in combination with TALEN and Cas9 systems to provide high-throughput cell line engineering anywhere in the genome.
How the PinPoint system works
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 two distinct approaches: i) the PinPoint-FC system that involves the well-characterized phiC31 integrase system or ii) the PinPoint-HR
system that uses either TALE-Nuclease or Cas9 genome engineering tools to induce a double-stranded DNA break in the genome and insertion of the
PinPoint placement site by homology-directed recombination (HDR) in a site-specific manner.
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 involves 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.
Comparison of Cell Line Engineering Technologies
|Overview:||Use the PhiC31 integrase and PhiC31 donor vectors to make stable cell lines.||Use the PhiC31 system to place the PinPoint site in a target genome, then use the PinPoint integrase and PinPoint donor vectors to create isogenic stable cell lines at the PinPoint locus every time.||Use TALEN or Cas9 nucleases & an HR donor vector to place the PinPoint site at an exact target locus anywhere in the genome, then use the PinPoint integrase and PinPoint donor vectors to create isogenic stable cell lines at the same PinPoint locus every time.||Use TALEN or Cas9 nucleases & an HR donor vector for stable Knock-In applications.|
|Most convenient for:||Fast stable cell line generation||Efficient, isogenic stable cell line generation; controlling for integration site variation.||Efficient, isogenic stable cell line generation at any genomic location; controlling for integration site variation.||Cell line engineering at any genomic location, when only a single engineered line is needed. Also ideal for Knock-Out & Gene Correction applications|
|# of Transfections required:||
|Ease of Protocol||Very Easy||Easy||More Difficult for Placement, then Easy for Donor Integration||More Difficult|
|Efficiency||High||High||Low for Placement, then High for Donor Integration||Low|
|Easily Create Multiple Lines Targeting the Same Location||No||Yes||Yes||No|
|Intensive screening required||No||No||Only once to generate Platform cell lines||Yes, for every new stable cell line|
|Unlimited Cargo Size||Yes||Yes||Yes||No|