uC31 integrase, derived from Streptomyces phage uC31, is a site-specific recombinase, equally functional in mammalian cells [1,2]. It has also been reported that uC31 integrase can mediate the integration of a vector bearing an attB sequence into endogenous sequences in mammalian genomes, resulting in an inserted vector sequence flanked by two hybrid junctions; attR and attL [2]. Genomic sites specifically targeted by the attB sequence are named pseudo attP sites, and contain a sequence partially identical to the phage attP site [2]. Unlike the widely used Cre and FLP recombinases that can catalyze both integration and excision, uC31 integrase-mediated integration is unidirectional [1,3]. Furthermore, the integration mediated by uC31 integrase requires no cofactor, and results in the long-term expression of foreign genes. These advantages make it an attractive tool for genomic engineering. The uC31 integrase-mediated recombination system is increasingly applied to the modification of somatic cells for genomic function research, ex vivo manipulation of embryonic stem cells to reconstruct genetically modified animals, and the in vivo correction of mutant genes for therapeutic purposes [4][5][6][7][8]. uC31 integrase, a site-specific recombinase, can effectively mediate foreign genes bearing an attB sequence integrated into pseudo attP sites. We have previously identified two pseudo attP sites, BpsF1 and BpsM1 from the bovine genome. In this study, two new pseudo attP sites, BF4 and BF10, were discovered using half-nested inverse PCR from cow fibroblasts. The genomic locations of these two pseudo attP sites were identified by direct sequencing and a BLAST search, and it was confirmed that they reside at positions 4q31 and 10q35 by fluorescence in situ hybridization analysis. Subsequently, the distinct integration frequencies of the four pseudo attP sites were examined. The BF4 site was identified as a hotspot where sitespecific integration occurred in most of the cell clones examined, accounting for 74% (42 ⁄ 57) of the integration; much more than the integration frequency for BF10 (7%; 4 ⁄ 57), BpsF1 (7%; 4 ⁄ 57) and BpsM1 (0 ⁄ 57). Interestingly, similar to other hotspots identified in the human and mouse genomes, in which transgenes integrated at hotspots result in high expression, the GFP gene integrated at hotspot BF4 was expressed at high levels in cow fibroblasts, as confirmed by fluorescence microscopy and FACS analysis. Furthermore, ELISA showed that the expression level of the GFP gene integrated at the BF4 site averaged 328 lgAEmg )1 , which is more than twofold higher than that integrated at the BF10 site. This study suggests that somatic cells carrying a desired gene integrated at the BF4 site can be used as nuclear donors to generate valuable transgenic animals by nuclear transfer.Abbreviations GFP, green fluorescence protein; SCNT, somatic cell nuclear transfer.