The Analysis And Application Of Integration Sites Mediated By?C31 Integrase In Bovine Genome

?C31 integrase, derived from Streptomyces phage?C31, was found to be one type of site-specific recombinases, which can efficiently mediate the site-specific integration of plasmid bearing an 34bp attB site into endogeneous sequence in mammalian genomes, and the integration sites being targeted is named pseudo attP site, partial identity with phage attP site. Pseudo attP sites have been detected in various species, and the data show that there exist multiple pseudo attP sites in the genomes. It is reported that 31, 57, 3, 3, 3 pseudo attP sites are found from human, mouse, rat, rabbit, drosophila genomes, respectively. We previously also identified two pseudo attP sites from bovine genome, named BpsM1?BpsF1 respectively. For more efficient construction of transgenic cow with the mediation of?C31 integrase, it is urgent to identify pseudo attP site which is efficiently targeted and even facilitate transgene expression. In the present study, we extent our previous work to find more pseudo attP sites in bovine genome and characterize them.Firstly, we applied inverse PCR (iPCR) to amplify the integration sites from two GFP gene-stably integrated cell clones driven by?C31 integrase. The results showed that the attB core sequences of inserting vector were broken, and recombinated with bovine genome in these two clones. It indicated that the recombination event occurred as a site specific manner, and the chromosome sites which were targeted are pseudo attP sites. BLAST analysis showed that these two pseudo attP sites resided at chromosome 4 and chromosome 10, named BF4, BF10, respectively. The analysis of genomic context of the two pseudo attP sites suggested that neither of them be at coding regions. The fluorescence in suit hybrydization (FISH) analysis further demonstrated that BF4 site was defined at chromosome 4q31, and the BF10 site was located at chromosome 10q35.Secondly we determined the integration frequency in bovine genome and the expression profile of the transgene which was integrated at different pseudo attP sites. The results showed that 42 of 57 (74%) clones in which GFP genes were integration at BF4 site, while the number of cell clones of integration at BF10 was only 4, accounting for 7% (4/57) of the integration events. The integration frequency of the BpsF1 was also 7% (4/57), which was as the same as that of BF10. No clone was detected as integrating at BpsM1 site. The remaining clones could not be detected as integrated at any of the four sites. These data strongly suggested that the integration sites mediated by?C31 integrase be significantly different from random integration and apparently preferred for BP4 site, which we therefore called it an integration hotspot. Furthermore ELISA and Western blot were used to assay the expression level of the transgenes. The results showed that the expression level of GFP gene integrated at BF4 is 328?g/mg, over 2-fold higher than that integrated at BF10(135?g/mg). These findings are similar to the results which are observed in human, mouse genomes. Transgene could express with high level when it is integrated at hotspot. Thus we have identified a pseudo attP site BF4, which not only can be targeted most frequently, but also facilitate the expression of inserted gene.Finally, we explored the possibility of using a combination of?C31 integrase and homologous recombination mechanism to increase the frequency of integration into the targeted site. We added the BF4 or BF10 sequence to a GFP gene expressed vector to construct an inserted recombination vector, then an attB sequence was introduced to the linear sits. After cotransfected with the?C31 integrase expression plasmid (pCMV-Int) into cow fibroblast, and integration sites were analyzed for the individual clones, we find that the integration rate of modified vectors were significant increased, up to 20-50%. However, The expression of the transgenes were as the same as unmodified vector.Our studies are of a significant value for production of transgenic animals by somatic cell nuclear transfer (SCNT) technique. The strategy provides us with an effective selection of donor cells. The cells that transgene was inserted at BF4 site can be readly used as donor cells for producing transgene animals by SCNT approach. These pave the way to generating transgenic farm animals with high efficiency, time-saving and lower cost.