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Figure 4 | BMC Biotechnology

Figure 4

From: Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome

Figure 4

Comparing the activity of fifteen serine recombinases in human HT1080 cells and in mouse ES cells. A. HT1080 cells containing a single integrated attP array CCAG HyTK attB array reporter construct were stably transfected with an integrase expression plasmid selecting for antibiotic resistance. The transfected clones were then subjected to gancyclovir selection to identify those that had lost the CCAG HyTK marker gene and then screened for recombinant attR sites to identify those that had undergone integrase-mediated site-specific deletion of CCAG HyTK. The two bars shown for each integrase reflect the results of two independent experiments. (Additional file 1: Table S4). None of the other eight integrases yielded recombinant gancyclovir resistant clones and they are not shown in this part of the figure. B; ES cells containing a attP array CCAG HyTK attB array reporter integrated at the ROSA26 locus were transiently co-transfected with an integrase expression plasmid and a linearized PGKneo construct to normalize for differences in the efficiency of transfection. Gancyclovir resistant clones were screened for attR sites. Two experiments were carried out and the bars reflect the means of the normalized activities (Additional file 1: Table S5). The analyses were carried out twice on a single attP array CCAG HyTK attB array reporter cell line and thus the data is shown as the mean of the two experiments. C; HT1080 cells that had been transfected with the empty expression vector (CCAG iresZeo) or with the indicated integrase expression vector were transiently transfected with the attP array CCAG HyTK attB array reporter and scored for site-specific recombination by PCR. For φ370 and TG1 integrases the integrase expression vector was co-transfected with the reporter construct, the remaining experiments were carried out with HT1080 cell lines that had been stably transfected with the respective integrase expression construct.

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