Fig 8. Retrohoming frequencies of Ll.LtrB variants containing positively selected mutations identified by PacBio sequencing.

The prominent mutations in EBS1 (G282A) and DIVb (position 642) were combined with other positively selected mutations that showed covariation and tested for retrohoming into a plasmid target site in HEK-293 cells in culture medium supplemented with 80 mM MgCl₂. Retrohoming frequencies were measured at 24 h after transfection of the expression plasmids in adherent HEK-293 cells by Taqman qPCR assays of the 5’- and 3’-integration junctions relative to the hyg ^R marker adjacent to the target site. The bar graphs show retrohoming frequencies of the variants relative to that of the wild-type intron. Values with error bars are the mean ± SD for at least three experimental trials. Values without error bars were tested once. The negative control WT(-) is the wild-type intron tested without additional MgCl₂ in the culture medium. Retrohoming frequencies for the wild-type intron measured by Taqman qPCR of 3’-integration junctions ranged from 0.06–0.11% in this series of experiments.