System Biosciences
PinPoint-FC System for Platform Cell Line Generation & Retargeting (includes PIN300A-1, FC200PA-1, PIN200A-1, PIN510A-1, & PIN600A-1)
- SKU:
- PIN300A-KIT
- Availability:
- Usually Shipped in 5 Working Days
- Size:
- 1 Kit
- Shipping Temperature:
- RT/Blue Ice/ Dry Ice
Description
PinPoint-FC System for Platform Cell Line Generation & Retargeting (includes PIN300A-1, FC200PA-1, PIN200A-1, PIN510A-1, & PIN600A-1). Cat# PIN300A-KIT. Supplier: SBI System Biosciences
- The PinPoint-FC attP Placement Vector (# PIN300A-1)
- The PhiC31 Integrase (# FC200PA-1)
- The PinPoint Integrase Expression Plasmid (# PIN200A-1)
- The CAG PinPoint Donor Vector (# PIN510A-1)
- The CAG-GFP-T2A-Luc PinPoint Positive Control Donor Vector (# PIN600A-1)
Products
Overview
Making cell line generation and retargeting reliable, efficient, and amenable to high-throughput production, the PinPoint-FC System for Platform Cell Line Generation and Retargeting provides all the plasmids you need for implementing the PinPoint System using the PhiC31 Integrase to place the PinPoint attP site into the target genome.
The PinPoint-FC System includes:
- The PinPoint-FC attP Placement Vector (# PIN300A-1)
- The PhiC31 Integrase (# FC200PA-1)
- The PinPoint Integrase Expression Plasmid (# PIN200A-1)
- The CAG PinPoint Donor Vector (# PIN510A-1)
- The CAG-GFP-T2A-Luc PinPoint Positive Control Donor Vector (# PIN600A-1)
With the PinPoint Targeted Integration System, you can ensure that only a single copy of your transgene is inserted into the genome.
- Create isogenic cell lines
- Achieve high-efficiency integration with no insert size-limit
- Turn cell line construction into a high-throughput process
How It Works
Targeted integration with the PinPoint System
Engineering cells with the PinPoint Targeted Integration System is a two-step process:
- Insertion of the PinPoint placement site (PinPoint attP) into the target genome using either the PhiC31 Integrase, which preferentially inserts into transcriptionally active sites of the genome, or the CRISPR/Cas9 System for insertion into a defined locus, such as the AAVS1 Safe Harbor Site.
- Integration of a PinPoint Vector into the pre-placed attP site. Integration is mediated by the PinPoint Integrase, which mediates a recombination event between the PinPoint attB site (located on the PinPoint Vector) and the PinPoint attP site (placed into the target genome in step 1). Because the PinPoint Integrase only recognizes your pre-placed PinPoint attP site, you get very stringent control of integration location.
A third optional step involves removal of extra vector sequences using the Cre/Lox system, leaving behind only your expression cassette and a single LoxP site.
Placing the PinPoint attP with the PhiC31 Integrase System
If you choose to use the PinPoint-FC attP Placement Vector to insert the PinPoint attP site, the PGK promoter on the Placement Vector combines with the promoterless puromycin marker on any of SBI’s PinPoint Donor Vectors, to enable puromycin selection only if the PinPoint Donor Vector integrates at the correct site.
Supporting Data
See the PinPoint Targeted Integration System in action
Figure 1. Efficient placement of the PinPoint attP site using the PhiC31 System. Step 1 of the PinPoint Targeted Integration System—the PinPoint attP site was placed into HEK293 cells using the PinPoint-FC Placement Vector (Cat.# PIN300A-1) and the PhiC31 Integrase (Cat.# FC200PA-1). Positive cells were selected with G418 (neomycin resistance) for fourteen days and six separate lines were picked and expanded. After eleven days, the cells were fixed and stained with a solution of 50% methanol plus 1% methylene blue. The plates were washed twice with PBS and allowed to air dry. Only the plate of cells that were transfected with the PhiC31 Integrase Expression Plasmid showed a robust number of cells.