PAGE 5 OF 14 Nucleic Acids Research, 2005, Vol. 33, No. 22 e189 Figure 3. Assaying the accuracy of integration promoted by the fC31 integrase in DT40 cells. (A) schematic representation of a fC31 integrase promoted site- specific integration of the promoterless fC31 attPneo trapping plasmid into the CCAG attB HyTk gene within the mini-chromosome A9 CCAG attB HyTk. (B) PCR assay for the accuracy of the integration promoted by the fC31 integrase was established using four primers which flanked asymmetrically both the attB and attP sites and the product attL and attR sites. The diagram indicates the respective positions of the different primer combinations. (C) 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 attB HyTk and expressing the fC31 integrase with fC31attPneo. The controls used in this experiment were extracts of DT40 cells doped with plasmids CCAG attB HyTk and fC31 attPneo to single copy levels. Details of the PCR are given in Materials and Methods and in Supplementary Data. vertebrate cells and allowed expression of the integrase gene as a bi-cistronic mRNA. We then introduced the linearized expression vector into DT40 cells containing the attB-modi- ﬁed mini-chromosome, selected for stably transfected clones and analysed expression of the integrase gene by western blotting (data not shown). The results indicated that the integ- rase comprise 0.01% of the total protein in the respective cell extracts. We also used immunocytochemistry to conﬁrm that the protein did not enter the nucleus unless modiﬁed appro- priately (data not shown). In order to assay the activity of the integrase in the DT40 cells we assembled a plasmid, fC31 attPneo (Figure 2B), that contains an attP site for the fC31 integrase 50 to the coding region of a promoterless G418 resistance gene. The plasmid fC31 attPneo was then transfected by electroporation into DT40 cells containing the attB-modiﬁed mini-chromosome and expressing either the native or nuclear localization signal modiﬁed fC31 integrase. Transfectants were recovered in 96-well dishes and G418 selection applied. No G418 res- istant clones were recovered from the clone (Table 1) express- ing the native integrase indicating that the background of integration of the attPneo into the host genome is undetect- able. The two clones expressing integrase tagged with the nuclear localization signal each gave G418 resistant clones at a frequency of 10 4 per electroporated cell (Table 1). In these experiments the proteins tagged at the N- and C- termini with nuclear localization signals were equally efﬁcient in promoting integration reactions (transfections 89 and 98). These results therefore do not conﬁrm those of others (20) who observed that the protein tagged at the C-terminus functions about three times more efﬁciently than that tagged at the N- terminus. These differences may reﬂect the nature of the cell lines used in our respective experiments. We analysed the products of the transfections of 2.5.01 and 4.5.01 by PCR (Figure 3). The attL and attR sites were detected in 81 out of 96 G418 resistant clones. In the other 15 clones one or other or both of the two anticipated products were absent (e.g. clones 43, 49, 52, 82, 85 and 95 as numbered from the top left), the clone contained only an attB site (clones 65 and 77) and had presumably escaped selection or the PCR products were ambiguously sized (clones 30, 46, 52, 58, 60, 62 and 88). The clones giving rise to these incorrect products were not analysed further but their existence indicates the need to con- ﬁrm by molecular techniques that the products of any selection are as intended. We sequenced the attR and attL sites of two clones (1 and 7) as representative of the majority and found that both were precisely as predicted for accurate site-speciﬁc recombination. Overall the results of this analysis indicate that the integrase promotes accurate site-speciﬁc recombination in chicken DT40 cells. The ﬁdelity of the plasmid integration reaction into the mini-chromosome was conﬁrmed by gel elec- trophoresis, ﬁlter transfer and hybridization for two such clones. The data on one of these clones are illustrated in Supplementary Figure 1.