The transgene was determined to have integrated at a single site, precisely at 85.72 Mb, on murine chromosome 5, region E-1 (NT109320.4), inside an intergenic region, NCBI Build m37.1 (Fig. 1B).
No tumors taken from mice given active φC31 integrase and the luciferase donor plasmid were found to have integrated at a preferred pseudo attP site in the mouse genome, even though one or two out of nineteen might have been expected to be positive by random chance.
A single band was detected upon restriction digestion of the genomic DNA by the enzymes HindIII and EcoRI, suggesting that the transgene had integrated at a single locus, as the transgene contained no restriction sites targeted by the enzymes (Figure 2A, left panel).
FISH analysis confirmed that the pHRT construct had integrated at a telomere (clone 22, Fig. 6A).
PCR analysis of transformants indicated that a single copy of RFP-hygMX had integrated at MATa.
In clone 1.4, the pHRT construct had integrated at the telomere of the q-arm of a large marker chromosome.
Several Tetr transformants were obtained and analysed by colony PCR, which indeed verified that the individual pORI19-derivative had integrated at the expected chromosomal location.
Southern blotting (Fig. 3B) and PCR analysis (data not shown) of genomic DNA from the double-resistant cells confirmed that both constructs had integrated at the expected locus and that all endogenous copies of TbAGO1 had been deleted.
The new retrotransposed copies were all found to have integrated at short poly-A sequences, varying in length between 4 and 21 consecutive A residues, with 25 of 30 being between 9 and 15 bp (inclusive) in length.
The clones in which pHRT integration had occurred at a telomere were able to produce RFP after a variable number of cell divisions, whereas the clone in which pHRT had integrated at an interstitial site was never able to express RFP.
