e189 Nucleic Acids Research, 2005, Vol. 33, No. 22 We wanted to establish how the efﬁciency of the integrase reaction varied as a function of cell number and the amount of DNA in the transfection. We therefore took one cell line (A9- CCAGattBHyk fC31–50NLS2) expressing the fC31 integrase tagged at the N-terminus and carried out two further series of transfections (numbered 39–41 and 42–44) in which we varied each of these parameters with one of the cell lines termed NLS2. The results of these experiments (Table 1) demonstrated that the number of recovered clones increased in proportion both to the amount of DNA and the number of electroporated cells and therefore extend the observations of others who have used the fC31 integrase in a different context (21). The results in Table 1 also indicate the reproducibility of the reaction; reactions carried out within days of one another are reproducible to within a factor of two but the efﬁciency varies more over longer intervals. Our earlier reactions (2.5.01 and 4.5.01) were 4-fold more efﬁcient than those carried out later. We are not sure why this is so. There may be variations in the quality of the fC31 attPneo DNA used in the transfec- tion, the cell culture medium or in the epigenetic status of the target DNA. Despite this relatively small variation the results demonstrate that the fC31 integrase along with its cognate attachment sites provides a robust and reliable tool for integ- rating DNA into vertebrate and other genomes. Site-specific integration of large DNA fragments cloned in a BAC vector using fC31 integrase We wanted to use the fC31 integrase for integration of large fragments of DNA into deﬁned loci so we next built a BAC vector, Apra fC31attPneo/pBAC3e (Figure 2C) containing the attPneo trapping cassette (Figure 2B). We conﬁrmed by PCR and by gel electrophoresis and ﬁlter hybridization that this vector integrated in site speciﬁcally (Supplementary Figure 1). In order to investigate the ability of the fC31 integ- rase to integrate large fragments of DNA we needed a suitable test sequence. Alphoid DNA is a tandemly repeated sequence found at the centromere of human chromosomes and has been shown to be mediate centromere function. We chose this as our test sequence for two reasons. First, it is of low sequence complexity and so it has a sequence organization that is easy to analyse; this is a particularly important when one wishes to establish whether a sequence has been integrated intact. Second, tandemly repeated sequences are particularly prone to re-arrangement and thus if we were able to integrate this sequence intact then we could argue conﬁdently that one should be able to use the system to integrate a sequence more typical of vertebrate genomic DNA. We cloned four differ- ently sized segments of Y chromosome alphoid DNA (Figure 4A) of 33, 70, 110 and 140 kb into a BAC vector Apra fC31attPneo/pBAC3e and transfected them into the DT40 cell line A9-CCAGattBHyTK fC31–50NLS2. The efﬁ- ciency varied between experiments as would be expected given variations in the quality of DNA cloned in low copy number vectors (Table 2) and were lower than with the attP- neo plasmid. We analysed the G418r clones recovered after transfection for the presence of the cloned insert by pulsed- ﬁeld gel electrophoresis (PFGE), blotting and PCR for the recombinant attL site (Figure 4C and D and Supplementary Figure 2). In interpreting the data in Figure 4 it is important to PAGE 6 OF 14 know that the 49B(A)A9 mini-chromosome (19), derived as it is, from the human Y chromosome contains alphoid DNA similar in sequence to that cloned in the BAC vector, Apra fC31attPneo/pBAC3e and which consequently hybridizes to the probe used to detect the sequence we are integrating into the chromosome. This sequence that we are introducing into the chromosome by site-speciﬁc recombination therefore appears in addition to pre-existing chromosomal fragments of 15 and 90 kb that are present on the native 49B(A)A9 mini-chromosome and are detected on the blots shown in Figure 4B. These blots of the transfected clones demonstrated that we could recover clones in which the DNA had integrated intact (lanes denoted as N in Figure 4C). These blots also show a change in size of the 15 kb alphoid fragment present on the native chromosome in those clones in which the BAC has integrated. This change in fragment size arises because the 15 kb alphoid fragment is present on the same SacI fragment as the integration site (for the positions of these two sequences see Figure 1). Site-speciﬁc integration introduces both the alphoid DNA insert and the BAC vector and thereby intro- duces a new SacI site immediately adjacent to the 15 kb alphoid DNA. This leads to the change in size of the smaller fragment. Not surprisingly the efﬁciency with which we recovered clones with intact BAC insert declined with the size of the insert so that with the 140 kb BAC we were only able to recover intact BAC in 10% of the G418r clones (Figure 4C and Table 2). The G418r clones which did not contain the intact insert were found to be of two types (indic- ated above the lanes in Figure 4C). Type 1 re-arrangements were often mixtures of inserts of two different sizes. These clones contained both recombinant attL and attR sites (Figure 4D). This type of clone was predominant in the case of the 70 kb BAC. We suggest that these re- arrangements arose as a result of the incoming BAC being nicked or otherwise damaged on one strand and that this dam- age was repaired by gene conversion at DNA replication from the undamaged sister chromatid. We suggest that the deletions in the products of the repair arise as a result of the gene conversion events occurring between tandemly repeated arrays that were aligned out of register. In some cases the BAC was detectably repaired from the alphoid DNA already present on the A9 mini-chromosome as judged by a deletion in this DNA; these events are referred to as 1* and one of these is shown in the panel illustrating the data obtained with the 140 kb BAC. The type II events contained no detectable insert but are G418r. PCR and ﬁlter hybridization analysis showed that these clones were deleted for sequences ﬂanking the expected attL and attR sites (Figure 4D). These clones may have arisen as a result of a double-strand break in the CCAG- fCattB-HyTk gene being resected and repaired by ligation to the neo coding sequence native to the 49B(A)A9 mini- chromosome. Alternatively they may have arisen by site- speciﬁc integration of a BAC with a double-strand break being resected prior to a similar non-homologous repair pro- cess. Since we see few such clones in the control transfections where we do not transfect with BAC DNA we consider the second explanation more likely but have no way of proving this interpretation. The goal of our project and of others is to be able to integrate DNA intact rather than re- arranged and so we did not characterize these re-arrangements in any more detail.