Assaying the accuracy of integration promoted by the φC31 integrase in DT40 cells

Copyright information:Taken from "Iterative assembly of large and complex transgenes by combining the activities of φC31 integrase and Cre recombinase"Nucleic Acids Research 2005;33(22):e189-e189.Published online 15 Dec 2005PMCID:PMC1316120.© The Author 2005. Published by Oxford University Press. All rights reserved () schematic representation of a φC31 integrase promoted site-specific integration of the promoterless φC31 neo trapping plasmid into the CCAG HyTk gene within the mini-chromosome A9 CCAG HyTk. () PCR assay for the accuracy of the integration promoted by the φC31 integrase was established using four primers which flanked asymmetrically both the and sites and the product and sites. The diagram indicates the respective positions of the different primer combinations. () The results of the PCR assay when applied to 96 clones isolated as resistant to G418 following transfection of DT40 cells containing the mini-chromosome A9 CCAG HyTk and expressing the φC31 integrase with φC31neo. The controls used in this experiment were extracts of DT40 cells doped with plasmids CCAG HyTk and φC31 neo to single copy levels. Details of the PCR are given in Materials and Methods and in Supplementary Data.