e189 Nucleic Acids Research, 2005, Vol. 33, No. 22 PAGE 12 OF 14 Figure 8. ISSI of large fragments molecules into mini-chromosome A9 CCAG loxP HyTk attP in DT40 cells. (A) Illustrates the iterations of plasmid and large fragment integration reactions into the CCAG loxP HyTK attP site present in the starting cell line ISSI 2 and analysed in (C–E). The cell line ISSI 2 was transfected with the plasmid attBloxPBSRattB to yield cell line 267 as described in Figure 7. This cell line was then transfected sequentially with a BAC containing 70 kb of Y alphoid DNA cloned in fC31BAC/PCCAGloxPHyTkP to yield cell line 293 and then with a PAC containing 80 kb of X alphoid DNA cloned in fC31PAC/ BloxPBSRB to yield cell line 419. The recombination sites analysed by PCR in section E below are indicated. (B) Pulsed-field gel analysis of 80 kb X and 70 kbY alphoid DNA cloned in the BAC and PAC vectors used to transfect the cells as described in A. The DNA was digested with NotI, which excises the insert, prior to gel electrophoresis. The image is of an ethidium bromide stained gel. (C) PFGE and filter hybridization analysis of the intact modified mini-chromosomes in cell lines 293 and 419. The control is the mini-chromosome isolated from the cell line A9-CCAGattBHyk fC31-50NLS2 (abbreviated NLS2) modified with CCAG attB HyTk and expressing the fC31 integrase. This filter transfers were probed with Y and X alphoid DNA sequences and illustrate the incremental increase in size of the mini- chromosome that follows from the introduction of the 70 and 80 kb alphoid Y and X alphoid DNA sequences. (D) Restriction enzyme mapping of the integrated alphoid DNA sequences. DNA extracted from the indicated cell lines was digested with either BglII or BclI, size fractionated by PFGE, filter transferred and the transfer probed with either X or Y alphoid DNA sequences. BglII but not BclI cuts the integrated DNA sequences at the junctions between the insert and vector. The BglII digests therefore identify the DNA sequences that have been introduced into the chromosome while the co-hybridization of the 170 kb fragment in the BclI digest of the 419 cell line establishes linkage between the Y and X alphoid DNA sequences as predicted in (A). (E) Identification by PCR of the predicted junctions indicated in (A).