General method for the modification of different BAC types and the rapid generation of BAC transgenic mice.

TitleGeneral method for the modification of different BAC types and the rapid generation of BAC transgenic mice.
Publication TypeJournal Article
Year of Publication2004
AuthorsSparwasser T, Gong S, Li JYH, Eberl G
Date Published2004 Jan
KeywordsAnimals, Animals, Genetically Modified, Chromosomes, Artificial, Bacterial, Cloning, Molecular, Escherichia coli Proteins, Gene Library, Gene Targeting, Genetic Engineering, Genetic Vectors, Mice, Mice, Transgenic, Rec A Recombinases, Recombination, Genetic, Transformation, Genetic, Transgenes

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.

Alternate JournalGenesis
PubMed ID14755803
Grant ListF32 NS041702 / NS / NINDS NIH HHS / United States
F32 NS041702-02 / NS / NINDS NIH HHS / United States