PAGE 3 OF 14 Transfection Cell line 4.8.02 2.5.01 4.5.01 39 40 41 42 43 44 89 98 304 A9-CCAGattBHyk fc31 integrase A9-CCAGattBHyk fc31-50NLS1 A9-CCAGattBHyk fc31-50NLS2 A9-CCAGattBHyk fc31-50NLS2 A9-CCAGattBHyk fc31-50NLS2 A9-CCAGattBHyk fc31-50NLS2 A9-CCAGattBHyk fc31-50NLS2 A9-CCAGattBHyk fc31-50NLS2 A9-CCAGattBHyk fc31-50NLS2 A9-CCAGattBHyk fc31-30NLS2 A9-CCAGattBHyk fc31-30NLS2 288-ISSI Table 1. Efficiency of site-specific integration of plasmid DNA promoted by fC31 integrase alone and in conjunction with Cre recombinase (ISSI) Nucleic Acids Research, 2005, Vol. 33, No. 22 e189 Transfected plasmid attPneo attPneo attPneo attPneo attPneo attPneo attPneo attPneo attPneo attPneo attPneo BBloxPBSR Amount (mg) Number of cells transfected % plated out Number of stably transfected colonies 50 100 100 25 250 25 25 250 25 100 25 20 5.7 · 107 3.2 · 107 3.2 · 107 2.34 · 107 2.34 · 107 2.45 · 108 1.24 · 107 1.24 · 107 1.24 · 108 2 · 107 3.8 · 107 5 · 107 100 100 100 25 25 25 25 25 25 25 25 25 0 2912 3920 90 379 389 31 398 315 433 152 226 Absolute efficiency <2 · 10 8 9.1 · 10 5 12.2 · 10 5 1.53 · 10 5 6.47 · 10 5 0.66 · 10 5 0.96 · 10 5 12.8 · 10 5 1.01 · 10 5 8.66 · 10 5 1.6 · 10 5 1.8 · 10 5 Table 2. Assaying the efficiency of integration of BAC DNA promoted by the fC31 integrase alone and during ISSI into a mini-chromosome DT40 cells Transfection number 19 51 72 53 419 Cell line BAC 50NLS2 50NLS2 50NLS2 50NLS2 ISSI 293 33 kb a attPneoBAC 70 kb a attPneoBAC 110 kb a attPneoBAC 140 kb a attPneoBAC 80 kbX a BBLoxPBSR BAC Amount mg 48 190 520 88 500 Number of cells transfected % plated out 2.0 · 108 2.0 · 108 2.75 · 108 2.0 · 108 5.7 · 108 100 100 100 100 100 Resistant clones 41 1078 158 288 >2000 Number of clones analysed 20 39 39 39 20 No intact 13 20 7 4 4 CCAG HyTk constructs containing attachment or loxP sites to the mini-chromosomes was detected by digestion with SacI, hybridization with an alphoid DNA probe and the replacement of a 20 kb SacI fragment by one of 17 kb in size. The site-speciﬁc integration experiments were carried out as fol- lows: cells were electroporated in the usual conditions with the indicated amount of DNA and immediately after transfection were plated out in 96-well dishes. Selection was applied 18 h later. DT40 colonies were counted after 12–14 days and counts were corrected using the Poisson distribution for the small underestimate arising from the random distribution of trans- fected cells into a limited number of wells. The efﬁciencies given in Tables 1 and 2 are the total number of resistant colonies recovered divided by the total number of cells elec- troporated. We refer to this ﬁgure as the absolute efﬁciency of integration in the table headings and text. PCR and filter hybridization analysis Conventional agarose and pulsed-ﬁeld gels were as described previously (19). Filter hybridization was as described (19). The sequences of the primers used in the PCR to check the breakpoints in the fC31 integration reactions and in the ISSI reaction sequence (Figure 7) are given in the Supplementary Data (Table 1). The markers used in Figure 4C were the mid- range I PFG set from New England Biolabs. The sizes of the informative marker molecules are 15, 33, 48.5, 63.5, 82, 97, 112, 130.5, 145.5 and 160.5 kb. The conditions for the PCR in Figures 3 and 4 and Supplementary Figure 2 (attL) are as follows 35 cycles: 92 C for 20 s, 62 C for 20 s and 72 C for 30 s. The buffer used was a 16 mM (NH4)2SO4, 67 mM Tris–HCl, pH 8.8, 0.1% Tween-20, 1 mM MgCl2 and the Taq polymerase was from Bioline. The primer sequences used are given in Supplementary Table 1. The attB primers were used at 400 nM and the attP primers at 200 nM ﬁnal concentrations. Markers used in the PCR in Figure 4 were the 100 bp ladder from NEB. RESULTS fC31 integrase in DT40 cells We initiated our experiments by studying the ability of the fC31 integrase (11) to promote site-speciﬁc integration of a closed circular plasmid into a genomic locus. In order to do this we ﬁrst of all used sequence targeting to introduce an attB site into a deﬁned position on a human mini-chromosome contained in a DT40 hybrid cell line. The attB site was placed between the CCAG promoter (18) and the coding region of the hygromycin–thymidine kinase (17) fusion gene. The mini- chromosome used in these experiments was the 49B(A)A9 mini-chromosome (Figure 1A) that was derived from the human Y chromosome and is described in detail elsewhere (19). This mini-chromosome is largely composed of DYZ1,2 repeated sequences but contains two arrays of alphoid DNA at one end of the chromosome. These are 15 and 90 kb in size. The attB targeting construct disrupted the G418 resistance gene at the left hand end of the chromosome (Figure 1B) (19). Next we wanted to express the fC31 integrase in the cells containing the modiﬁed mini-chromosome. The fC31 integrase is 68 kDa in molecular weight and is of bacterial origin. It therefore seemed likely that it would need a nuclear localization signal to function in our experiments. Therefore we introduced sequences encoding either the native fC31 integrase or the integrase tagged at either the N- or C-termini with the SV40 virus large T antigen nuclear localization signal into an expression vector, CCAG IRES zeo (Figure 2A) that conferred zeocin resistance upon