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Figure 5 | BMC Biotechnology

Figure 5

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 5

Comparing the activity of seven different serine recombinases in human HT1080 cells for their utility in recombinase mediated cassette exchange. A. Cell lines containing a single integrated attP array CCAG HyTK attP array reporter construct and stably expressing the indicated integrase were transfected with the attB array CCAG neo attB array integration reporter construct using lipofectamine. The experiment was carried out three times using two independent cell lines for each of the seven integrases. The number of G418-resistant clones generated by the cassette exchange reaction promoted by the different integrases was normalized by transfection using lipofectamine with a uniform amount of linearized CCAG neo plasmid. Open bars correspond to the number of colonies generated with the reporter plasmid divided by the number of colonies generated with the linearized CCAG neo (for the raw data see Additional file 1: Table S6). Between seven and ten colonies of each transfection were picked and assayed for site-specific recombination by PCR and the total yield of colonies generated by site-specific recombination is represented by the filled bars. B. Cell lines that had been stably transfected with the indicated integrase expression construct were transiently transfected with the indicated integration reporter construct and after three days assayed for site specific recombination by PCR. Two different reactions were used for the assay; one for the Bxb1 integrase and the other for the remaining integrases. The gel on the final panel shows the PCR reaction products obtained when the indicated reporter constructs were transfected into HT1080 cells that expressed no integrase and assayed for site-specific recombination by either of the two reactions.

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