title: Establishment of a rapid and scalable gene expression system in livestock by site-specific integration.
abstract: Somatic cell-mediated transgenesis is routinely used to transfer exogenous genes to livestock genomes. However, transgene insertion events are essentially random which may lead to transgene silencing or alter animal phenotype because of insertional mutagenesis. To overcome these problems, we established a gene manipulation system in goat somatic cells based on homologous recombination and flp recombinase-mediated site-specific integration. First, we performed gene targeting to introduce an frt-docking site into the α1 (I) procollagen (ColA1) locus in goat somatic cells. Second, the targeted cell clones were rejuvenated by embryo cloning, and the vigorous cells with targeted frt were reestablished. Third, a gene-replacement system was used to introduce an EGFP reporter gene into the targeted ColA1 locus via flp mediated recombination. As a result, the transgenic somatic cell exhibited faithful expression of EGFP gene under control of the CMV promoter. Similarly, other expression vectors can be introduced into the defined site to evaluate gene functions or express valuable proteins. The gene manipulation system described here will be applicable in other livestock somatic cells, and would allow for the rapid generation of livestock with transgene targeted to the defined site.
authors.affiliation: Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, PR China.
authors.forename: Huiqing
authors.initials: H
authors.lastname: Yu
authors.forename: Xuebin
authors.initials: X
authors.lastname: Wang
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
authors.forename: Zhuzi
authors.initials: Z
authors.lastname: He
authors.forename: Guohui
authors.initials: G
authors.lastname: Liu
authors.forename: Xujun
authors.initials: X
authors.lastname: Xu
authors.forename: Jianquan
authors.initials: J
authors.lastname: Chen
authors.forename: Guoxiang
authors.initials: G
authors.lastname: Cheng
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002478:Cells, Cultured; D003001:Cloning, Molecular; D024042:Collagen Type I / Q000235:genetics; D046149:Embryo Culture Techniques; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D006041:Goats / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D058751:Livestock / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D024042:Collagen Type I; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins