TAILIEUCHUNG - Báo cáo khoa học: A uC31 integrase-mediated integration hotspot in favor of transgene expression exists in the bovine genome

uC31 integrase, a site-specific recombinase, can effectively mediate foreign genes bearing anattBsequence integrated into pseudoattPsites. We have previously identified two pseudoattP sites, BpsF1 and BpsM1 from the bovine genome. In this study, two new pseudoattPsites, BF4 and BF10, were discovered using half-nested inverse PCR from cow fibroblasts. | ỊFEBS Journal A pC31 integrase-mediated integration hotspot in favor of transgene expression exists in the bovine genome Hai-Long Ou Ying Huang Li-Juan Qu Miao Xu Jing-Bin Yan Zhao-Rui Ren Shu-Zhen Huang and Yi-Tao Zeng Shanghai Institute of MedicalGenetics Shanghai Jiao Tong University China Keywords bovine genome integration hotspot pseudo attP site transgenic animal uC31 integrase Correspondence . Zeng Shanghai Institute of Medical Genetics Shanghai Children s Hospital 24 1400 West Beijing Road Shanghai 200040 China Fax 86 21 6247 5476 Tel 86 21 6279 0545 E-mail ytzeng@ Received 22 September 2008 revised 23 October 2008 accepted 28 October 2008 doi 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

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