PinPoint site placement and targeting

Pinpoint-FC attP placement
The PinPoint attP site was efficiently placed into HEK293 cells or HeLa cells. Approximately 8x10^5 cells were transfected with 40 ng PinPoint-FC placement vector (Cat# PIN300A-1) and 1,960 ng phiC31 integrase expression vector (Cat# PIN200A-1) at a 1:50 ratio, respectively. Positive cells were selected with 400 ug/ml G418 for 14 days. Six separate lines of HEK293 and HeLa cells were picked and expanded. After 11 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. The number of visible colonies were imaged and colony number counts assessed.

Neomycin resistant HEK293 cells with placed PinPoint site

These lines were then transfected with a control PinPoint Gene Donor vector (PIN600A-1) for precise integration. Unlike the R4 integrase used in a similar system, the PinPoint integrase does not recognize pseudo-sites and will only integrate at its placed recognition sequence. This feature results in the increased efficiency of correctly retargeted cell lines compared to the R4 integrase system. The entire therapeutic gene expression cassette is active and fully insulated with cHS4 DNA elements.

Neomycin resistant HEK293 cells with placed PinPoint site

To verify that the PinPoint donor construct targeted to the correct site, junction PCR can be employed to amplify across the PinPoint integrase recombination site (attL) junction. Primers are designed within the placed puromycin marker cassette and within the PGK promoter are used in nested PCR reactions to verify the correct PinPoint integration structure. Three separate HEK293 colonies that were GFP positive were expanded for 3 days with subsequent genomic DNA isolation. Approximately 50 ng of genomic DNA was used in the nested PCR reactions and the products separated on 2% agarose gels and stained with ethidium bromide before imaging. The results clearly indiavcte the predicted, PinPoint integrase-mediated insertion of the PIN600a-1 donor vector at the placed attP site with high precision.