Extended Data Fig. 8: Recombination activity of D7278-Zif268 in human cells.
From: Activation of recombinases at specific DNA loci by zinc-finger domain insertions
a Recombination activity of D7278-Zif268 and D7-ZF on the genomic loxF8-zif reporter, carrying the Zif268 motifs flanking the loxF8 target site in HEK293TloxF8-zif cells two days after mRNA transfections. Schematic representation of the unrecombined and recombined genomic loxF8-zif reporter is shown to the left. Primers used for PCR to detect the recombination of the reporter are depicted in grey. The distance between the loxF8-5-zif(B) target sites and the size of the possible PCR products is indicated. An agarose gel image of PCR products generated using the indicated primers to detect reporter recombination is shown to the right. The HEK293TloxF8-zif cells treated with GFP mRNA only were used as a wild-type control. The upper band represents the unrecombined product (line with two triangles), and the lower band represents the recombined product (line with one triangle); M = Marker. b Inversion efficiency of D7278-Zif268 and D7-ZF on the endogenous loxF8 locus in HEK293TloxF8-zif cells two days post mRNA transfection. Schematic representation of a fraction of the F8 gene, displaying the two possible orientations of the loxF8 locus (wild-type and inverted orientation) is shown to the left (adapted from Lansing et al.8). Primers used for PCR to detect the orientation of the locus are displayed as green, blue, and red arrows. The position of the loxF8 sites and the distance between them are indicated. An agarose gel image of PCR products generated using the indicated primer combinations to detect the orientation of the loxF8 locus of the HEK293TloxF8-zif cells is shown to the right. The HEK293TloxF8-zif cells treated with GFP mRNA only were used as a wild-type control. A PCR reaction on DNA isolated from iPSCs derived from a patient carrying the int1h inversion was used as an inversion control. Primer combinations are indicated. M = Marker. The assays were performed three times (n = 3 biologically independent replicates).
