title: Transgenesis of Tol2-mediated seamlessly constructed BAC mammary gland expression vectors in Mus musculus.
abstract: Bacterial artificial chromosomes (BACs) are vectors that are capable of carrying gene fragments of up to 300 kb in size, and in theory, harbor cis-regulatory elements that are necessary for the expression of specific genes. Therefore, BACs can effectively alleviate or even eliminate the position effect induced by gene-integration, rendering these as ideal expression vectors of exogenous genes. However, the number of relevant studies involving BACs as vectors of exogenous genes are limited. In the present study, we converted the BAC regulatory region of the Mus musculus Wap gene into a mammary gland-specific expression vector. Using the galK-based positive-negative selection method, we seamlessly replaced the Wap gene in a BAC with Homo sapiens GPX3, MT2, and Luc genes while keeping the original mammary gland-specific regulatory sequence intact, without introducing any extra sequences (Loxp/Frt). To improve the efficiency of creating BAC transgenic mice, we used a Tol2 transposon system optimized for mammalian codons and eliminated 100 kb of sequence from the BAC 5' end (173 kb), which resulted in an 8.5% rate of successful gene transmission via pronuclear injection. The results of the present study indicate that seamlessly constructed BAC expression vectors can be used for the transmission of the GPX3 gene.
authors.affiliation: School of Life Sciences,Henan University, Kaifeng 475004, PR China.
authors.forename: Yaohui
authors.initials: Y
authors.lastname: Yang
authors.affiliation: School of Life Sciences,Henan University, Kaifeng 475004, PR China.
authors.forename: Wenyuan
authors.initials: W
authors.lastname: Wang
authors.affiliation: School of Life Sciences,Henan University, Kaifeng 475004, PR China.
authors.forename: Tian
authors.initials: T
authors.lastname: Huang
authors.affiliation: School of Life Sciences,Henan University, Kaifeng 475004, PR China.
authors.forename: Weimin
authors.initials: W
authors.lastname: Ruan
authors.affiliation: School of Life Sciences,Henan University, Kaifeng 475004, PR China; Institute of Bioengineering, Henan University, Kaifeng 475004, PR China. Electronic address: gscao@henu.edu.cn.
authors.forename: Gengsheng
authors.initials: G
authors.lastname: Cao
mesh_terms: D000818:Animals; D022202:Chromosomes, Artificial, Bacterial; D004251:DNA Transposable Elements; D005260:Female; D017353:Gene Deletion; D018014:Gene Transfer Techniques; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D008321:Mammary Glands, Animal / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic
chemical_list: D004251:DNA Transposable Elements; C486484:GPX3 protein, human; C116888:Gpx3 protein, mouse; D005979:Glutathione Peroxidase
keywords: Bacteria artificial chromosome; Glutathione peroxidase; Tol2-transposon; Transgenic mice; galK positive and negative selection