Transposon-mediated BAC transgenesis in zebrafish and mice
1 Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
2 Division of Population Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
3 Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), 1111 Yata, Mishima, Shizuoka 411-8540, Japan
BMC Genomics 2009, 10:477 doi:10.1186/1471-2164-10-477Published: 16 October 2009
Bacterial artificial chromosomes (BACs) are among the most widely used tools for studies of gene regulation and function in model vertebrates, yet methods for predictable delivery of BAC transgenes to the genome are currently limited. This is because BAC transgenes are usually microinjected as naked DNA into fertilized eggs and are known to integrate as multi-copy concatamers in the genome. Although conventional methods for BAC transgenesis have been very fruitful, complementary methods for generating single copy BAC integrations would be desirable for many applications.
We took advantage of the precise cut-and-paste behavior of a natural transposon, Tol2, to develop a new method for BAC transgenesis. In this new method, the minimal sequences of the Tol2 transposon were used to deliver precisely single copies of a ~70 kb BAC transgene to the zebrafish and mouse genomes. We mapped the BAC insertion sites in the genome by standard PCR methods and confirmed transposase-mediated integrations.
The Tol2 transposon has a surprisingly large cargo capacity that can be harnessed for BAC transgenesis. The precise delivery of single-copy BAC transgenes by Tol2 represents a useful complement to conventional BAC transgenesis, and could aid greatly in the production of transgenic fish and mice for genomics projects, especially those in which single-copy integrations are desired.