title: General method for the modification of different BAC types and the rapid generation of BAC transgenic mice.
abstract: Most genome projects have relied on the sequencing of bacterial artificial chromosomes (BACs), which encompass 100-300 kb of genomic DNA. As a consequence, several thousand BAC clones are now mapped to the human and mouse genome. It is therefore possible to identify in silico a BAC clone that carries a particular gene and obtain it commercially. Given the large size of BACs, most if not all regulatory sequences of a gene are present and can be used to direct faithful and tissue-specific expression of heterologous genes in vitro in cell cultures and in vivo in BAC-transgenic mice. We describe here an optimized and comprehensive protocol to select, modify, and purify BACs in order to generate BAC-transgenic mice. Importantly, this protocol includes a method to generate, within 2 days, complex plasmid cassettes required to modify BACs, and to efficiently modify different types of BACs selected from the two major BAC libraries available. Altogether, using a combination of genomic database analysis, overlap PCR cloning, and BAC recombination in bacteria, our approach allows for the rapid and reliable generation of "pseudo knockin" mice. genesis 38:39-50, 2004.
authors.affiliation: Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Munich, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Sparwasser
authors.forename: Shiaoching
authors.initials: S
authors.lastname: Gong
authors.forename: James Y H
authors.initials: JY
authors.lastname: Li
authors.forename: Gerard
authors.initials: G
authors.lastname: Eberl
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D029968:Escherichia coli Proteins / Q000235:genetics; D015723:Gene Library; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics; D011938:Rec A Recombinases / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D011938:Rec A Recombinases