Identification And Site-specific Modification Of The Safe Locus Rosa26 In Bovine

Continuous and stable expression of exogenous genes in vivo or in vitro is important for basic research and application of transgenic technologies.The traditional transgenic technique generally integrates exogenous genes into genome by random insertion.The integration site and the copy number can not be accurately controlled,thus leading to the unstable or differentially expression of the exogenous genes,which limit the application and development of transgenic technologies.In recent years,with the rapid development of site-specific integration technology based on homologous recombination technique,it is possible to integrate exogenous genes into a pre-selected locus with a single copy so as to overcome the defects of random integration.Therefore,find a safe locus suitable for insertion and efficient expression of exogenous genes is of great importance.Rosa26 is the most widely used "safe locus" in mammalian genome,which was first discovered by Friedrich and Soriano in mice.The study of Rosa26 found that this loci can support the expression of exogenous genes at all stages of embryonic development and in all tissues of adults without adverse effects.Now the targeted modification of Rosa26 in mice has been widely used in continuous and conditional expression of exogenous genes.These modification result in the establishment of several hundred mouse models,which play important roles in the basic study such as gene fuction?disease models and drug development research.After the discovery of Rosa26 in mice,the Rosa26 locus of human,rat,pig,rabbit and sheep were continually determined through comparative study.This loci were highly conserved in sequence,and supported stable and high expression of exogenous genes in all species.Bovine is an important agricultural economy animal,the current research focus on genetic modification to let it produce higher quality milk?safer animal products and has more resistant to disease.However there have no study about "safe locus" in bovine until now.In this study we identified the bovine Rosa26(bRosa26)locus,and attempted to establish a gene specific target system in this locus.In this study,the mouse Rosa26 promoter and exon I sequences were used as a template to search the bovine genome database.A highly conserved sequence(sequence similarity 83%)on bovine chromosome 22 was found as the putative bRosa26 locus.This region contains both the bRosa26 gene and the synteny genes among different species.The sequence of mouse,rat,human and bovine Rosa26 showed conservation of>75%similarity.The bRosa26 exon I was predicted according to multiple sequence alignment result.The 3' RACE and 5' RACE were employed to determine the fully transcription of the bRosa26 gene.RT-PCR and Q-PCR assay demonstrated that bRosa26 gene was expressed in a wide variety of adult tissues.To determine whether the bRosa26 promoter can drive gene expression ubiquitously like other species,we constructed and transiently transfected a bRosa26 promoter-driven EGFP transgene into different cell lines.A high level of EGFP expression was detected which suggested that the bRosa26 locus is an ideal site for ubiquitous expression of exogenous genes.In ordet to generate the transgenic bovines with stable and ubiquitous expression of exogenous genes,we attempted to introduce a Cre-dependent EGFP reporter gene into the bRosa26 locus by TALENs technology.We then achieved an efficient recombination in cells mediated by TAT-Cre protein.A long term high expression of the EGFP reporter was detected in the recombinant cells which confirmed that bRosa26can habour the exogenous genes' stable expression.Using somatic cell nuclear transfer technology,we successfully generated bRosa26 gene-targeting fetuses and bovines.Using RT-PCR?Q-PCR?Western Blot assay,EGFP was found to be stable and widespread in all stages of embryonic development and in all tissues of adults.The insertion did not significantly interfere with the expression of neighboring genes.Taken together,these data strongly suggested that the bRosa26 locus is an ideal site for ubiquitous expression of exogenous genes.In this study,a fetal fibroblast line was successfully established,and a pair of heterologous loxP sites were introduced into bRosa26,which can be used for gene replacement based on a recombinase mediated cassette exchange(RMCE).Using it,we efficiently replaced the green fluorescent protein EGFP reporter gene with the red fluorescent protein tdDIMER gene.This proved that the bRosa26 could mediate the efficient RMCE.Finally,we efficiently integrated three targeted vector into the bRosa26,in which EGFP was driven by CAG?EFla and PGK promoter respectively.In the three cell lines,EGFP was stable expressed at different times,and its expression did not significantly interfere with the expression of neighboring genes,and the CAG promoter has the highest activity to activate the expression of EGFP,when compared with the bRosa26?EF1a and PGK promoter.In summary,we have for the first time identified and characterized the bovine Rosa26 locus.We further certificated that the bRosa26 locus could support the exogenous genes' expression ubiquitously.More importantly,the RMCE technology combining the TAT-Cre protein could be used to produce transgenic bovine stably expressing any gene of interest at the bRosa26 locus.It will serve as an excellent platform for generating genetically modified bovines for biomedical and agricultural applications.